KR101313975B1 - Use of effectors of glutaminyl and glutamate cyclases - Google Patents

Abstract

The present invention can be treated by a novel physiological substrate of glutaminyl cyclase (QC, BC 2.3.2.5) of mammals, a novel effector of QC and the use of said effector, and modulation of QC-activity, For example, a disease selected from the group consisting of duodenal cancer, colorectal cancer, Zolliger-Ellison syndrome, family history British dementia (FBD), and family history Danish dementia (FDD) caused by or without Helicobacter pylori infection It provides a pharmaceutical composition comprising the effector for treating.

Description

Use of effectors of glutaminyl and glutamate cyclases

The present invention provides an intramolecular cyclization of an N-terminal glutamine residue to pyroglutamic acid (5-oxo-proline, pGlu ^* ) under ammonia liberation, and an N-terminal glutamate residue into pyroglutamic acid under the release of water. It relates to glutaminyl cyclase (QC, EC 2.3. 2.5), which catalyzes the cyclizing action within.

The present invention identifies mammalian QC as a metallase, provides a novel physiological substrate of QC in mammals, and provides a novel anti-QC effector, a use of the QC effector, and a condition that can be treated by modulating QC-activity. It provides a pharmaceutical composition comprising a QC effector for treatment. It also reveals that metal interactions are a useful approach to the development of QC inhibitors.

In a preferred embodiment, the present invention is directed to using a QC activity effector for the treatment or alleviation of a condition treatable by modulation of QC- and DP IV- activity in combination with an inhibitor of DP IV or DP IV-like enzymes. to provide.

It also provides a screening method for identifying and selecting QC active effectors.

Glutaminyl cyclase (QC, EC 2.3. 2.5) catalyzes the intramolecular cyclization of the ^{N-terminal glutamine residue with pyroglutamic acid (pGlu *) while releasing ammonia.} The first tropical plant Carica papaya (Carica papaya) was first developed by Messer in 1963. papaya ) (Messer, M. 1963 Nature 4874, 1299). After 24 years, the corresponding enzymatic activity was found in the animal pituitary gland (Busby, WHJ et al. 1987 J Biol Chem 262, 8532-8536; Fischer, WH and Spiess, J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). . For mammalian QC, the conversion of Gln to pGlu by QC can be indicated for the precursors of TRH and GnRH (Busby, WHJ et al. 1987 J Biol Chem 262, 8532-8536; Fischer, WH and Spiess, J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). In addition, in the initial position measurement experiment of QC, it was found that in addition to improving the function estimated in the synthesis of peptide hormones, it coexisted with the estimated catalytic product in the pituitary gland of cattle (Bockers, TM et al. 1995 J Neuroendocrinol 7, 445-453). On the other hand, the physiological function of plant QC is less clear. In the case of enzymes derived from C. papaya, its role in plant defense mechanisms against pathogenic microorganisms has been suggested (El Moussaoui, A. et al. 2001 Cell Mol Life Sci 58, 556-570). QCs deduced from other plants were recently identified by sequence comparison (Dahl, SW et al. 2000 Protein Expr Purif 20, 27-36). However, the physiological actions of these enzymes are still unclear.

QCs known from plants and animals show the correct specificity for L-glutamine at the N-terminal position of the substrate, and their kinetic behavior was found to follow the Michaelis-Menten equation (Pohl. , T. et al. 1991 Proc Natl Acad Sci USA 88, 10059-10063; Consalvo, AP et al. 1988 Anal Biochem 175, 131-138; Gololobov, MY et al. 1996 Biol Chem Hoppe Seyler 377, 395-398). The primary structure of C. papaya-derived QCs and mammal-derived highly conserved QCs were compared, but no sequence homology was revealed (Dahl, SW et al. 2000 Protein Expr Purif 20,27-36). Plant QCs have been shown to belong to a novel family of enzymes (Dahl, SW et al. 2000 Protein Expr Purif 20, 27-36), whereas mammalian QCs have been shown to have positive sequence homology with bacterial aminopeptidase. Appearing (Bateman, RC et al. 2001 Biochemistry 40,11246-11250), it comes to the conclusion that QCs derived from plants and animals have different evolutionary origins.

EP 02 011 349.4 discloses polynucleotides encoding insect glutaminyl cyclase and polypeptides encoded thereby. In addition, the above patent application provides a host cell comprising an expression vector containing the polynucleotide of the present invention. Isolated polypeptides and host cells comprising insect QC are useful in methods for screening agents that reduce glutaminyl cyclase activity. Such agents are described as useful as pesticides.

Alzheimer's disease (AD) is characterized by an abnormal accumulation of extracellular amyloid plaques closely associated with dystrophic neurons, reactive astrocytes and microglia (Terry, RD and Katzman, R. 1983). Ann Neurol 14, 497-506; Glenner, GG and Wong, CW 1984 Biochem Biophys Res Comm 120, 885-890; Intagaki, S. et al. 1989 J Neuroimmunol 24, 173-182; Funato, H. et al. 1998 Am J. Pathol 152, 983-992; Selkoe, DJ 2001 Physiol Rev 81, 741-766). Amyloid-β (Aβ) peptide is a primary component of senile plaque and is believed to be directly involved in the phathogenesis and progression of AD, a hypothesis supported by genetic studies (Glenner, GG and Wong, CW. 1984 Biochem Biophys Res Comm 120, 885-890; Borchelt, DR et al. 1996 Neuron 17, 1005-1013; Lemere, CA et al. 1996 Nat Med 2, 1146-1150; Mann, DM and lwatsubo, T. 1996 Neurodegeneration 5, 115-120; Citron, M. et al. 1997 Nat Med 3,67-72; Selkoe, DJ 2001 Physiol Rev 81, 741-766). Aβ is produced by the proteolytic process of β-amyloid precursor protein (APP) (Kang, J. et al. 1987 Nature 325, 733-736; Selkoe, DJ 1998 Trends Cell Biol 8, 447-453). , Which are subsequently cleaved sequentially by β-secretase at the N-terminus of Aβ and by γ-secretase at the C-terminus of Aβ (Haass, C. and Selkoe, DJ 1993 Cell 75 , 1039-1042; Simons, M. et al. 1996 J Neurosci 16 899-908). In addition to the main Aβ peptide (Aβ1-42/40) starting with L-Asp at the N-terminus, many heterologous forms truncated at the N-terminus occur in senile plaques. These shortened peptides are neurotoxic in vitro and have been reported to aggregate more rapidly than full-length isoforms (Pike, CJ et al. 1995 J Biol Chem 270 23895-23898). N-resected peptides are overproduced in early onset family history AD (FAD) subjects (Saido, TC et al. 1995 Neuron 14, 457-466; Russo, C. et al. 2000 Nature 405,531-532), Down syndrome (DS) Early in the brain and increasing with age (Russo, C. et al. 1997 FEBS Lett 409,411-416, Russo, C. et al. 2001 Neurobiol Dis 8, 173-180; Tekirian, TL et al. 1998 J Neuropathol Exp Neurol 57, 76-94). Finally, their quantity reflects the degree of disease progression (Russo, C. et al. 1997 FEBS Lett 409, 411-416). An additional post-translation process can further alter the N-terminus by isomerizing or racemizing the aspartate at positions 1 and 7 and cyclizing the glutamate at residues 3 and 11. The pyroglutamate-containing isoforms [pGlu ³ ]Aβ(3-40/42) at position 3 represent the predominant form of the N-resected species in senile plaques-about 50% of the total Aβ amount-(Mori, H. 1992 J Biol Chem 267, 17082-17086, Saido, TC et al. 1995 Neuron 14, 457-466; Russo, C. et al. 1997 FEBS Lett 409, 411-416; Tekirian, TL et al. 1998 J Neuropathol Exp Neurol 57, 76-94; Geddes, JW et al. 1999 Neurobiol Aging 20,75-79; Harigaya, Y. et al. 2000 Biochem Biophys Res Commun 276,422-427), they are also present in pre-amyloid lesions (Lalowski , M. et al. 1996 J Biol Chem 271, 33623-33631). The accumulation of [pGlu ³ ]Aβ(3-40/42) peptide appears to be due to structural alterations that enhance aggregation and confer resistance to most amino-peptidase (Saido, TC et al. 1995 Neuron 14, 457-). 466; Tekirian, TL et al. 1999 J Neurochem 73,1584-1589). This evidence provides ^{clues as to the central role of the [pGlu 3} ]Aβ(3-40/42) peptide in the pathogenesis of AD. However, relatively little is known about their neurotoxicity and aggregation properties (He, W. and Barrow, CJ 1999 Biochemistry 38, 10871-10877; Tekirian, TL et al. 1999 J Neurochem 73, 1584-1589). Moreover, the action of these isoforms on glial cells and the responses of glial cells to these peptides are fully known, although activated glial cells (glia) are clearly associated with senile plaques and will actively contribute to the accumulation of amyloid deposits. There is not. In a recent study, the toxicity, aggregation properties and catabolism of Aβ(1-42), Aβ(1-40), [pGlu ³ ]Aβ(3-42) and [pGlu ³ ]Aβ(3-40) peptides were investigated. It was investigated in the culture of neurons and glial cells, and it was found that pyroglutamate modification worsens the toxicity of Aβ-peptide and also inhibits degradation by cultured astrocytes. ^{Shirotani et al. (2002) generated the [pGlu 3} ]Aβ peptide in vitro in primary conical neurons infected by the Sindbis virus. They constructed the amyloid precursor protein complementary DNAs encoding potential precursors for ^{[pGlu 3} ]Aβ by amino acid substitutions and deletions. For one artificial precursor starting with the N-terminal glutamine residue instead of glutamate in the wild precursor, natural conversion to pyroglutamate or enzymatic conversion by glutaminyl cyclase has been shown. [pGlu ³ ] The mechanism of cyclization of the N-terminal glutamate at position 3 in the natural precursor of Aβ was not determined in vivo (Shirotani, K., Tsubuki, S., Lee, HJ, Maruyama, K. ., and Saido, TC (2002) Neurosci Lett 327, 25-28).

Family history British dementia (FBD) and family history Danish dementia (FDD) are predominantly onset autosomal dominant diseases that are considered progressively recognized disorders as convulsions and cerebellar dysfunction (Ghiso, J. et. al. 2000, Ann. NY Acad Sci 903 , 129-137; Vidal, R. et al. 1999, Nature 399, 776-781; Vidal, R. et al. 2004, J Neuropathol Ezp Neurol 63 , 787-800). Similar to Alzheimer's disease, widespread flexible tissue and vascular amyloid deposits are formed in patients by hippocampal neurodegeneration, complement and glial activity (Rostagno, A. et al. 2002, J Biol). Chem 277 , 49782-49790). The disease is caused by different mutations in the BRI gene (SwissProt Q9Y287), so that the open reading frame (ORF) is 11 amino acids longer than the native BRI. In the case of FBD, the change in ORF is caused by a mutation in the BRI stop codon (BRI-L), whereas in FDD, the overlap-insertion of 10 nucleotides results in a larger BRI (BRI-D) (Ghiso J. et al. 2001 Amyloid 8 , 277-284; Rostagno, A. et al. 2002 J Biol Chem 277, 49782-49790). The transmembrane protein of BRI, category 2, is encoded on chromosome 13 and is shown to be processed by purines and other prohormone converting enzymes in the C-terminal region, releasing a long peptide of 23 amino acids (Kim, SH. et al. 2000 Ann NY Acad Sci 920 , 93-99; Kim SH et al. 2002 J Biol Chem 277, 1872-1877). The cleavage of the mutant BRI proteins BRI-D and BRI-L leads to the production of peptides (34 amino acids ABri and ADan), which produce amyloid fibril as well as non-fibrillar precipitates. Tend to aggregate (EI Agnaf, O. M et al. 2004 Protein Pept Lett 11, 207-212; EI Agnaf, OM et al. 2001 Biochemistry 40, 3449-3457; EI Agnaf, OM et al. 2001 J Mol Biol 310, 157-168; Srinivasan et al. 2003 J Mol Biol 333, 1003-1023). The ADan and ABri peptides have the same 22 amino acids at the N-terminus, but the C-terminal region is completely different. The C-terminal portion has been shown to be a portion required for fiber formation and neurotoxicity (EI Agnaf, OM et al. 2004 Protein). Pept Lett 11 , 207-212).

It has been known that the N-terminus of the ABri and ADan peptides is blocked by the formation of pyroglutamyl. In Alzheimer's disease, the formation of pyroglutamyl (pGlu) at the N-terminus of Aβ is formed from glutamic acid (Ghiso J. et al. 2001 Amyloid 8 ; Saido et al. 1996 Neuron 14 , 457-466). The formation of pyroglutamyl, in turn, causes the degradation of most of the amino peptidases, thereby immobilizing the peptides, leading to the progression of the disease. The formation of aggregates has been shown to proceed not only inside the secretory pathway of cells but also outside the cells (Kim et al. 2002 J Biol Chem 277, 1872-1877). Therefore, inhibition of pGlu formation at the N-terminus of neurotoxicity of ABri and ADan peptides by inhibition of glutamyl and glutamate cyclase represents a novel approach to the treatment of FBD and FDD.

Dipeptidyl peptidase IV (DP IV) is a post-proline (to a lesser extent, post-alanine, post-serine or post-glycine) found in various tissues within the body, including the kidney, liver and intestine. It is a cleavage serine protease and cleaves the N-terminal dipeptide from the peptide chain. Recently, it was found that DP IV plays an important role in neuropeptide metabolism, T-cell activation, cancer cell adhesion to epithelial tissue, and HIV entry into lymphoid cells (WO 02/34242, WO 02/34243, WO 03/002595 and WO 03/002596).

DP IV inhibitors described in WO 99/61431 include amino acid residues, thiazolidine groups or pyrrolidine groups, and salts thereof, in particular L- threo -isoleucyl thiazolidine, L- allo -isoleucyl thiazolidine, L- Threo -Isoleucyl pyrrolidine, L- Allo -Isoleucyl-thiazolidine, L- Allo -Isoleucyl pyrrolidine.

Further examples of low molecular weight dipeptidyl peptidase IV inhibitors include tetrahydroisoquinoline-3-carboxamide derivatives, N-substituted 2-cyanopyrroles and -pyrrolidines, N-(N'-substituted glycyl)- 2-cyanopyrrolidines, N-(substituted glycyl)-thiazolidines, N-(substituted glycyl)-4-cyanothiazolidines, amino-acyl-borono-prolyl-inhibitors, cyclopropyl -Such as fused pyrrolidines and heterocyclic compounds. Inhibitors of dipeptidyl peptidase IV are described in US 6,380,398, US 6,011,155; US 6,107,317; US 6,110,949; US 6,124,305; US 6,172,081; WO 95/15309, WO 99/61431, WO 99/67278, WO 99/67279, DE 198 34 591, WO 97/40832, DE 196 16 486 C 2, WO 98/19998, WO 00/07617, WO 99/ 38501, WO 99/46272, WO 99/38501, WO 01/68603, WO 01/40180, WO 01/81337, WO 01/81304, WO 01/55105, WO 02/02560 and WO 02/14271, WO 02/ 04610, WO 02/051836, WO 02/068420, WO 02/076450; WO 02/083128, WO 02/38541, WO 03/000180, WO 03/000181, WO 03/000250, WO 03/002530, WO 03/002531, WO 03/002553, WO 03/002593, WO 03/004496, It is described in WO 03/024942 and WO 03/024965, the teachings of which are incorporated herein by reference, in particular, in their entirety regarding such inhibitors, their definitions, uses and production.

Summary of the invention

The invention ^{Gln 1 -ABri, Gln 1 -ADan,} Gln 3 -Aβ (3-40 / 42), and Gln ¹ - gastrin (17 and 34) as a novel of QC in mammals, selected from the group consisting of Duodenal cancer, colorectal cancer, Zolliger-Ellison syndrome, which can be treated by physiological substrates, use of QC effectors, and modulation of QC activity, preferably due to or unrelated to Helicobacter pyroly infection. ), family history British dementia, and family history Danish dementia to provide a pharmaceutical composition comprising a QC effector for the treatment of a disease selected from the group consisting of.

Inhibition studies have shown that human QC is a metal-dependent transferase. QC apoenzymes can most effectively be reactivated by zinc ions, and the metal-binding motif of zinc-dependent aminopeptidase is also present in human QC. Compounds that interact with the active-site bound metal are potent inhibitors of QC.

Surprisingly, both recombinant human QC as well as QC-activators obtained from brain extracts catalyze the N-terminal glutaminyl as well as glutamate cyclization. The most surprising finding is that the cyclase-catalyzed Glu ¹ -conversion is advantageous around pH 6.0, while the Gin ¹ -conversion to the pGlu-derivative occurs optimally around pH 8.0. Therefore, since the formation of pGlu-Aβ-related peptides can be inhibited by inhibition of recombinant human QC and QC-activator obtained from porcine pituitary extract, according to the present invention, the enzyme QC is used to develop a drug for the treatment of Alzheimer's disease. It is the target of.

The present invention optionally comprises at least one QC effector in combination with a conventional carrier and/or excipient, or optionally at least one QC effector in combination with a conventional carrier and/or excipient and at least one DP IV-inhibitor. It provides a pharmaceutical composition for parenteral, enteral or oral administration comprising a

The present invention generally provides a QC-inhibiting agent represented by the following formula (1) or a pharmaceutically acceptable salt thereof including all stereoisomers.

In the above formula,

R ¹ to R ⁶ are each H or a branched or unbranched alkyl chain, a branched or unbranched alkenyl chain, a branched or unbranched alkynyl chain, carbocyclic, aryl, heteroaryl, heterocyclic , Aza-amino acids, amino acids or analogs thereof, peptides or analogs thereof; All of the above residues may be optionally substituted, and n may be 0 to 2.

The present invention

a) treatment of mammalian diseases that can be treated by modulating QC activity in vivo; And/or

b) Provides a glutaminyl cyclase (QC) effector for regulation of physiological processes, based on the action of pGlu-containing peptides triggered by regulation of QC activity.

Furthermore, the present invention is a compound that inhibits glutaminyl cyclase (QC, EC 2.3. 2.5) and/or QC-like enzymes in mammals, and QC activity inhibitors for treating pathogenic conditions associated with QC activity. Provides the use of.

The present invention also provides a new method of treatment of Alzheimer's disease and Down's syndrome.

The N-terminus of amyloid β-peptide deposited in the brain of Alzheimer's disease and Down's syndrome contains pyroglutamic acid. pGlu formation is important for the onset and progression of these diseases, as the modified amyloid β-peptide appears to exacerbate the onset and progression of the disease, with a stronger tendency for β-amyloid aggregation and toxicity (Russo, C. . Et al. 2002 J Neurochem 82, 1480-1489).

On the other hand, in wild Aβ-peptide (3-40/42), glutamic acid is present as the N-terminal amino acid. The enzymatic conversion of Glu to pGlu is not known to date. Moreover, natural cyclization from Glu-peptide to pGlu-peptide has not yet been observed. Therefore, one aspect of the present invention is to determine the role of QC in Alzheimer's disease and Down syndrome. This aspect is characterized by the synthesis of Aβ(3-11) and Aβ(1-11) comprising the amino acids glutamine instead of glutamic acid at position 3, QC, DP IV and DP IV-like enzymes, such modified for aminopeptidase. Characterized by the determination of the substrate properties of amyloid β-peptide, and the use of a QC inhibitor to prevent the formation of pGlu from the N-terminal glutaminyl residues of amyloid β-inducing peptides (1-11) and (3-11) do. The results are shown in Example 8. The applied method is described in Example 3.

To date, there has been no clue indicating that QC is involved in the progression of the disease, since glutamic acid is the N-terminal amino acid in Aβ (3-40/42, or 11-40/42). However, QC is the only known enzyme capable of forming pGlu at the N-terminus of a peptide. Another aspect of the present invention relates to the following research results and findings:

a) In addition to glutamine, QC catalyzes the cyclization of glutamic acid to pyroglutamic acid at a low rate,

b) Glutamic acid of APP or its subsequently formed amyloid-β-peptide is converted to glutamine in post-translational process by unknown enzymatic activity, and in the second step, QC is after processing of the amyloid β-peptide N-terminus Catalyzes the cyclization of glutamine to pyroglutamic acid,

c) Glutamic acid is converted to glutamine in a post-translational process by chemical catalysis or self-catalysis, and subsequently QC catalyzes the cyclization of glutamine to pyroglutamic acid after processing of the amyloid β-peptide N-terminus,

d) In the APP gene, which encodes the amyloid β-protein, there are mutations that cause Gln instead of Glu at position 3. After N-terminal translation and processing, QC catalyzes the cyclization of glutamine to pyroglutamic acid,

e) Due to the dysfunction of unknown enzymatic activity, glutamine is contained in the emerging peptide chain of APP, and sequentially QC is processed by the amyloid β-peptide N-terminus, followed by the N-terminal glutamine to pyroglutamic acid ring. Catalyzes anger.

QC is involved in the formation of pyroglutamic acid, which is a critical step in the five cases listed above, namely the aggregation of amyloid β-peptide. Thus, inhibition of QC leads to the onset and progression of Alzheimer's disease and Down syndrome, regardless of the mechanism by which cyclization occurs, the precipitation of plaque-forming Aβ (3-40/42) or Aβ (11-40/42). Can be prevented.

Glutamate is found at positions 3, 11 and 22 of the amyloid β-peptide. Among these, the mutation of glutamic acid (E) to glutamine (Q) at position 22 (corresponding to the amyloid precursor protein APP 693, Swissprot P05067) is described as a so-called Dutch type cerebroarterial amyloidosis mutation. come.

Β-amyloid peptides with pyroglutamic acid residues at positions 3, 11 and/or 22 are known to be more cytotoxic and hydrophobic than Aβ (1-40/42/43) (Saido TC 2000 Medical Hypotheses 54 (3): 427-429).

Multiple N-terminal variants were at different sites by β-site amyloid precursor protein-cleaving enzyme (BACE, β-site amyloid precursor protein-cleaving enzyme), a β-secretase enzyme (Huse JT et al. 2002 J. Biol Chem. 277 (18): 16278-16284), and/or aminopeptidase processing. In all cases, cyclization can take place according to a) to e) as described above.

^{To date, there was no evidence supporting the enzymatic conversion of the Glu 1} -peptide to pGlu-peptide corresponding to pathway a) by an unknown glutamyl cyclase (EC) (Garden, RW, Moroz, TP, Gleeson , JM, Floyd, PD, Li, LJ, Rubakhin, SS, and Sweedler, JV (1999) J Neurochem 72, 676-681; Hosoda R. et al. (1998) J Neuropathol Exp Neurol. 57,1089-1095). To date, such an enzymatic activity, capable of cyclizing ^{a Glu 1 -peptide carrying a negatively charged Glu 1} γ-carboxylate moiety under mild alkaline pH-conditions, protonated at the N-terminus, has not been identified. .

QC-activity against Gln ¹ -substrate decreases rapidly below pH 7.0. On the other hand, Glu ¹ -conversion seems to occur under acidic reaction conditions (Iwatsubo, T. et al. 1996 Am J Pathol 149, 1823-1830; Russo, C. et al. 1977 FEBS Lett 409, 411-416; Russo, C. et al. 2001 Neurobiol Dis 8, 173-180; Tekirian, TL et al. 1998 J Neuropathol Exp Neurol. 57, 76-94; Russo, C. et al. 2002 J Neurochem 82, 1480-1489; Hosoda, R. et al. 1998 J Neuropathol Exp Neurol. 57, 1089-1095; Garden, RW et al. 1998 J Neuropathol Exp Neurol. 56, 1089-1095; Garden, RW et al. 1999 J Neurochem 72, 676- 681).

In accordance with the present invention, it was investigated whether QC can recognize and alter amyloid-β inducible peptides under mild acidic conditions. Therefore, as a potential substrate for the enzyme, the peptides Gln ³ -Aβ(1-11)a, Aβ(3-11)a, Gln ³ -Aβ(3-11)a, Aβ(3-21)a, Gln ³ -Aβ(3-21)a and Gln ³ -Aβ(3-40) were synthesized and investigated. These sequences were chosen to mimic the ^{native N- and C-terminally truncated Glu 3} -Aβ peptides and Gln ³ -Aβ peptides, which may occur due to post-translational Glu-amideization.

In the present invention, papaya and human QC have been shown to catalyze both glutaminyl and glutamyl cyclization. Obviously, the primary physiological function of QC is to complete hormonal maturation in endocrine cells by glutamine cyclization before or during the hormone secretion process. Such secretory vesicles are known to have an acidic pH. Thus, the secondary activity of the enzyme in a narrow pH range of 5.0 to 7.0 may be the newly discovered glutamyl cyclase activity which also modifies the Glu-Aβ peptide. However, since Glu-cyclization occurs much slower than Gln-conversion, it is questionable whether glutamyl cyclization plays a significant physiological role. However, in the pathological findings of neurodegenerative diseases, the glutamyl cyclization is relevant.

By investigating the pH-dependence of this enzymatic reaction, the present inventors found ^{that the aprotonated N-terminus is essential for the cyclization of Gln 1} -peptide, and thus the pKa-value of the substrate is pKa- for QC-catalysis. It was found to be the same as the value (see Figure 17). Thus, QC stabilizes the intramolecular nucleophilic attack of the aprotonated α-amino moiety on the γ-carbonyl carbon that is electrophilically activated by amidation (Scheme 1).

Compared to the monovalent charge present on the N-terminal glutamine containing peptide, the N-terminal Glu-residue in the Glu-containing peptide is mainly divalently charged around neutral pH. Glutamate exhibits pKa-values of about 4.2 and 7.5, respectively, for the γ-carboxyl and α-amino moieties. That is, at neutral pH and above, even if the α-amino nitrogen is partially or wholly aprotonated and nucleophilic, the γ-carboxyl group is aprotonated and thus does not exhibit electrophilic carbonyl activity. . Therefore, intramolecular cyclization is not possible.

However, in the pH range of about 5.2 to 6.5, the two functional groups are, between their respective pKa-values, about 1 to 10% of the total N-terminal Glu-containing peptide (-NH ₂ ) or At concentrations of 10 to 1% (-COOH), both are present in non-ionized form. As a result, over the mildly acidic pH-range, the N-terminal Glu-peptides have both groups uncharged, and therefore, it is possible to stabilize the intermediate cyclization of the QC to the pGlu-peptide intramolecularly. That is, if the γ-carboxyl group is protonated, the carbonyl carbon becomes sufficiently electrophilic to allow nucleophilic attack by the aprotonated α-amino group. At this pH, the hydroxide ions act as a leaving group (Scheme 3). This assumption is confirmed by the pH-dependent data obtained for QC catalyzed conversion of Glu-βNA (see Example 11). In contrast to the glutamine conversion of Glu-βNA by QC, the optimum pH for catalysis is in the acidic range near pH 6.0, i.e., the substrate molecules with protonated γ-carboxyl groups and aprotonated α-amino groups simultaneously. It moves over many pH-ranges Furthermore, the kinematically determined pKa value of 7.55±0.02 corresponds exactly to that of the α-amino group of Glu-βNA (7.57±0.05) determined by titration.

Physiologically, the second order rate constant (or specificity constant, K _cat /K _M ) of QC-catalyzed glutamate cyclization at pH 6.0 can range 8,000 times slower than for glutamine cyclization (FIG. 17 ). . However, the non-enzymatic conversion of both model substrates Glu-βNA and Gln-βNA can be neglected and is consistent with the negligible pGlu-peptide formation observed in the present invention. ^{Thus, for pGlu-formation by QC, an acceleration coefficient of at least 10 8} can be obtained from the ratio of enzymatic to non-enzymatic rate constants (second order rate constants for enzyme catalysis and each Compared to the non-enzymatic cyclization first order rate constant, the catalytic proficiency factor is 10 ⁹ -10 ¹⁰ M ^-1 for Gln- and Glu-conversion, respectively). The conclusion from these data is that only enzymatic pathways that induce pGlu-forming in vivo can be conceived.

^{QC is very abundant in the brain, and given the recently discovered high conversion rate of 0.9 min -1} for the maturation of 30 μM of (Gln-)TRH-like peptide (Prokai, L., Prokai-Tatrai, K., Ouyang) , X., Kim, HS, Wu, WM, Zharikova, A., and Bodor, N. (1999) J Med Chem 42,4563-4571), about 100 hours for an appropriate glutamate-substrate if similar reaction conditions are given. The cyclization half-life of can be expected. Moreover, when brain QC/ECs are compartmentalized and localized in the secretory pathway, the actual in vivo enzyme and substrate concentration, and reaction conditions will be even more favorable for enzymatic cyclization in intact cells. And, if the N-terminal Glu is transformed into Gln, it is expected that pGlu-formation will be mediated much faster by QC. In vitro, both reactions were inhibited by applying inhibitors of QC/EC-activity (Figures 9, 10 and 15).

In summary, the present invention shows that a very large number of human QCs in the brain are catalysts for forming amyloid pGlu-Aβ peptides from Glu-Aβ and Gln-Aβ precursors that make up 50% or more of plaque deposits found in Alzheimer's disease. These findings reveal that QC/EC plays a role in the formation of senile plaques and thus a novel drug target for the treatment of Alzheimer's disease.

In a second embodiment of the invention, the amyloid β-derived peptides are substrates of dipeptidyl peptidase IV (DP IV) or DP IV-like enzymes, preferably dipeptidyl peptidase II (DP II). . DP IV, DP II or other DP IV-like enzymes produce amyloid β-peptide (3-11) having glutamine as the N-terminal amino acid residue, while the N-terminus of the modified amyloid β-peptide (1-11) Releases dipeptide from The results are shown in Example 8.

As described in the literature, prior to cleavage by DP II, DP IV or other DP IV-like enzymes, between aspartic acid (residue 1 of amyloid β-peptide) and alanine (residue 2 of amyloid β-peptide) Peptide bonds can be isomerized while forming isoaspartyl residues (Kuo, Y.-M., Emmerling, MR, Woods, AS, Cotter, RJ, Roher, AE (1997) BBRC 237, 188-191; Shimizu , T., Watanabe, A., Ogawara, M., Mori, H. and Shirasawa, T. (2000) Arch. Biochem. Biophys. 381,225-234).

These isoaspartyl residues make amyloid β-peptide resistant to aminopeptidase degradation, and consequently, the core plaques ^{contain a large amount of isoAsp 1} -amyloid β-peptide, which is converted at the N-terminus. Says decrease.

However, in the present invention, it is revealed for the ^{first time} that the N-terminal dipeptide H-isoAsp 1 -Ala ² -OH can be released by dipeptidyl peptidase, especially under acidic conditions. Furthermore, isomerization also occurs prior to cleavage by β-secretase, accelerating proteolytic processing, thus leading to the release of the N-terminal isoaspartyl bond of ^{isoAsp 1 -amyloid β-peptides, which subsequently} Was shown to be converted by DP II, DP IV or DP IV-like enzymes (Momand, J. and Clarke, S. (1987) Biochemistry 26, 7798-7805; Kuo, Y.-M., Emmerling, MR. , Woods, AS, Cotter, RJ, Roher, AE (1997) BBRC 237, 188-191). Thus, inhibition of isoaspartyl formation can reduce cleavage by β-secretase and then reduce the formation of amyloid β-peptides. ^{In addition, blocking isoAsp 1} -amyloid β-peptide conversion due to inhibition of DP II, DP IV or DP IV-like enzyme prevents the formation of ^{Glu 3} -Aβ into QC/EC-catalyzed [pGlu ^3]Aβ. I will be able to.

In a third embodiment of the present invention, a combination of inhibitors of DP IV-activity and inhibitors of QC can be used for the treatment of Alzheimer's disease and Down syndrome.

The combined effect of DP IV and/or DP IV-like enzyme and QC is illustrated below:

a) DP IV and/or DP IV-like enzymes cleave Aβ (1-40/42), dipeptide containing H-Asp-Ala-OH and Aβ (3-40/42) are released,

b) In the side reaction, QC catalyzes the cyclization of glutamic acid to pyroglutamic acid at a very low rate.

c) Glutamic acid is converted to glutamine by unknown enzymatic activity in a post-translational process at the N-terminus, and then QC catalyzes the cyclization of glutamine to pyroglutamic acid after processing of the amyloid β-peptide N-terminus.

d) Glutamic acid is converted to glutamine in a post-translational process by chemical catalysis or autocatalysis, and in the second step, QC catalyzes the cyclization of glutamine to pyroglutamic acid after amyloid β-peptide N-terminal processing.

e) There is a mutation in the APP gene encoding amyloid β-proteins, resulting in Gln instead of Glu at position 3 of Aβ, and after N-terminal translation and processing, QC catalyzes the cyclization of glutamine to pyroglutamic acid.

f) Glutamine is included in the new peptide chain of APP due to dysfunction of unknown enzymatic activity, and subsequently, QC loops glutamine at the N-terminus to pyroglutamic acid after processing of the N-terminus of amyloid β-peptide. Catalyzes anger.

Exposing the N-terminal Gln to QC-activity can also be initiated by different peptidase activities. Aminopeptidase makes it possible to sequentially remove Asp and Ala from the N-terminus of Aβ (1-40/42), thus revealing amino acid 3 that is liable to cyclize. Dipeptidyl peptidases such as DP I, DP II, DP IV, DP 8, DP 9 and DP 10 remove the dipeptide Asp-Ala at once. Thus, inhibition of aminopeptidase- or dipeptidylpeptidase-activity is useful for preventing Aβ (3-40/42) formation.

The combined effect of inhibitors of DP IV and/or DP IV-like enzymes and effectors of reducing QC activity is explained in the following manner:

a) Inhibitors of DP IV and/or DP IV-like enzymes inhibit the conversion of Aβ (l-40/42) to Aβ (3-40/42).

b) Thus, it is possible that the N-terminal exposure of glutamic acid is prevented and there is no conversion to glutamine, whether enzymatic or by chemical catalysis, which subsequently leads to pyroglutamic acid formation.

c) Inhibitors of QC also include any residual modified Aβ (3-40/42) molecules and pyroglutamic acid formation from those modified Aβ (3-40/42) molecules produced by mutations in the APP gene. Prevent it.

Within the present invention, a similar combined action of DP IV or DP IV-like enzyme and QC has been demonstrated for additional peptide hormone classes such as glucagon, CC chemokine and substrate P (substance P).

Glucagon is a 29-amino acid polypeptide secreted from pancreatic islet alpha-cells and acts to maintain euglycemia by stimulating hepatic glycogen decomposition reaction and glucose neolysis reaction (gluconeogenesis). Despite this importance, there is controversy over the mechanism responsible for the elimination of glucagon in the body. Pospisilik et al. investigated the enzymatic metabolism of glucagon using a delicate mass spectrometry technique to identify molecular products. Incubation of glucagon with purified porcine dipeptidyl peptidase IV (DP IV) resulted in successive production of glucagon (3-29) and glucagon (5-29). In human serum, glucagon was rapidly N-terminal cyclized after digestion with glucagon (3-29) and further DP IV-mediated hydrolysis was prevented. As a result of in vivo testing of glucagon after incubation with purified DP IV or normal rat serum, hyperglycemic activity was significantly lost, whereas similar incubation in DP IV-deficient rat serum resulted in any loss of glucagon bioactivity. Did not appear. As a result of measurement by mass spectrometry and bioassay, degradation was blocked by isoleucyl thiazolidine, a specific DP IV inhibitor. These results demonstrate that DP IV is the primary enzyme involved in the degradation and inactivation of glucagon. These findings have important implications for determining the concentration of glucagon in human plasma (Pospisilik et al., Regul Pept 2001 Jan 12; 96 (3): 133-41).

Human monocyte chemotactic protein (MCP-2) was originally isolated from stimulated osteosarcoma cells into chemokines produced together with MCP-1 and MCP-3. Von Coillie et al. (Van Coillie, E. et al. 1998 Biochemistry 37, 12672-12680) cloned the 5'-end extended MCP-2 cDNA from a human testicular cDNA library. It encodes an MCP-2 protein with 76 residues, but unlike the reported bone marrow-derived MCP-2 cDNA sequence, there was a difference in coding Lys instead of Gln at codon 46. This MCP-2Lys46 variant caused by single base polymorphism (SNP) was compared biologically with MCP-2Gln46. The coding region was sub-cloneed into the bacterial expression vector pHEN1 and transformed into Escherichia coli, and two MCP-2 protein variants were recovered in the periplasm between the cell membrane and the cell wall. As a result of Edman digestion, a Gln residue was found instead of pGlu at the _{NH 2 end.} rMCP-2Gln46 and rMCP-2Lys46, and the NH ₂ -terminal cyclic counterpart were tested on mononuclear cells by calcium mobilization and chemotaxis assays. It was observed that there was no significant difference in biological activity between the rMCP-2Gln46 and rMCP-2Lys46 isoforms. However, for both MCP-2 variants, NH ₂ -terminal pyroglutamate appeared to be essential for chemotaxis, but not for calcium mobilization. When cutting the ends, NH ₂ - - by the serine protease CD26 / dipeptidyl peptidase IV (CD26 / DPP IV) NH 2 of rMCP-2Lys46 but secretion terminal Gln-Pro dipeptide, the amino-synthesized having terminal pGlu MCP-2 remained unaffected. CD26/DPP IV-cleaved rMCP-2Lys46(3-76) appeared almost completely inactive in both chemotaxis and signal assays. These observations indicate that the NH ₂ -terminal pGlu in MCP-2 is essential for chemotactic activity, but also indicates that it protects the protein against degradation by CD26/DPP IV (van Coillie, E.. et al. Biochemistry. 1998 37,12672-80).

Within the present invention, it was observed that the formation of N-terminal pyroglutamate residues determined in glucagon (3-29) (Pospisilik et al., 2001) and MCP-2 isoform (van Coillie et al., 1998) is catalyzed by QC. /MS-analysis revealed.

In addition, after the N-terminal DP IV-catalyzed removal of the two peptides Lys-Pro and Arg-Pro from the substrate P, the remaining [Gln ⁵ ] substrate P 5-11 is [pGlu ⁵ ] substrate P 5 by QC. The conversion to 11 was demonstrated by LC/MS-analysis.

DP IV inhibitors are disclosed in WO 99/61431. In particular, amino acid residues and thiazolidine groups or pyrrolidine groups, and salts thereof, in particular L- threo -isoleucyl thiazolidine, L- allo -isoleucyl thiazolidine, L- threo -isoleucyl pyrrolidine, L DP IV inhibitors are disclosed, including- allo -isoleucyl thiazolidine, L- allo -isoleucyl pyrrolidine, and salts thereof.

Further examples of low molecular weight dipeptidyl peptidase IV inhibitors include tetrahydroisoquinoline-3-carboxamide derivatives, N-substituted 2-cyanopyrroles and -pyrrolidines, N-(N'-substituted glycyl). -2-cyanopyrrolidines, N-(substituted glycyl)-thiazolidines, N-(substituted glycyl)-4-cyanothiazolidines, amino-acyl-borono-prolyl-inhibitors, cyclo It is an agent such as propyl-fused pyrrolidines and heterocyclic compounds. Inhibitors of dipeptidyl peptidase IV are described in US 6,380,398, US 6,011,155; US 6,107,317; US 6,110,949; US 6,124,305; US 6,172,081; WO 95/15309, WO 99/61431, WO 99/67278, WO 99/67279, DE 198 34 591, WO 97/40832, DE 196 16 486 C 2, WO 98/19998, WO 00/07617, WO 99/ 38501, WO 99/46272, WO 99/38501, WO 01/68603, WO 01/40180, WO 01/81337, WO 01/81304, WO 01/55105, WO 02/02560 and WO 02/14271, WO 02/ 04610, WO 02/051836, WO 02/068420, WO 02/076450; WO 02/083128, WO 02/38541, WO 03/000180, WO 03/000181, WO 03/000250, WO 03/002530, WO 03/002531, WO 03/002553, WO 03/002593, WO 03/004496, WO 03/024942 and WO 03/024965, the teachings of which are incorporated herein by reference in their entirety, in particular with regard to such inhibitors, their definitions, uses and their production.

Preferred for use in combination with a QC effector are DP IV inhibitors, for example Hughes et al. NVP-DPP728A(1-[[[2-[{5-cyanopyridin-2-yl}amino]ethyl]amino]acetyl]-2-cyano-(S)-pi disclosed in 1999 Biochemistry 38 11597-11603 Rolledin) (Novartis), Hughes et al., eeting of the American Diabetes Association 2002, Abstract no. LAF-237(1-[(3-hydroxy-adamant-1-ylamino)-acetyl]-pyrrolidine-2(S)-carbonitrile) disclosed in 272 (Novartis); Yamada et al. 1998 Bioorg Med Chem Lett 8, TSL-225 (tryptophil-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) disclosed in 1537-1540, Asworth et al. 1996 Bioorg Med Chem Lett 6,, 1163-1166 and 2745-2748 disclosed 2-cyanopyrrolidides and 4-cyanopyrrolidides, Sudre et al. 2002 Diabetes 51, FE-999011 disclosed in 1461-1469 (Ferring), and a compound disclosed in WO 01/34594 (Guilford), and the dosage range set in the reference is applied.

More preferred DP IV inhibitors for use in combination with QC effectors are dipeptide compounds whose amino acids are selected from natural amino acids such as, for example, leucine, valine, glutamine, glutamic acid, proline, isoleucine, asparagine and aspartic acid. . The dipeptide-like compounds used according to the invention exhibit at least 10%, in particular at least 40%, the activity of plasma dipeptidyl peptidase IV or DP IV-like enzyme at a concentration (of dipeptide compound) of 10 μM. Decrease. Frequently, a decrease in activity of at least 60% or at least 70% is also desirable in vivo. Preferred compounds can also reduce the activity by up to 20% or 30%.

Preferred dipeptide compounds are N-valyl proryl, 0-benzoyl hydroxylamine, alanyl pyrrolidine, isoleucyl thiazolidine-like L- allo -isoleucyl thiazolidine, L- threo -isoleucyl pyrrolidine and Salts thereof, in particular fumar salts, and L- allo -isoleucyl pyrrolidine and salts thereof. Particularly preferred compounds are glutaminyl pyrrolidine and glutaminyl thiazolidine, H-Asn-pyrrolidine, H-Asn-thiazolidine, H-Asp-pyrrolidine, H-Asp-thiazolidine, H-Asp(NHOH)-pyrrolidine, H-Asp(NHOH)-thiazolidine, H-Glu-pyrrolidine, H-Glu-thiazolidine, H-Glu(NHOH)-pyrrolidine, H -Glu(NHOH)-thiazolidine, H-His-pyrrolidine, H-His-thiazolidine, H-Pro-pyrrolidine, H-Pro-thiazolidine, H-Ile-azididine, H -Ile-pyrrolidine, HL- Allo- Ile-thiazolidine, H-Val-pyrrolidine and H-Val-thiazolidine, and pharmaceutically acceptable salts thereof. Such compounds are disclosed in WO 99/61431 and EP 1 304 327.

Furthermore, the present invention provides the use of a QC effector in combination with a substrate-like peptide compound useful for competitive regulation of dipeptidyl peptidase IV catalysis. Preferred peptide compounds are 2-Amino octanoic acid -Pro-Ile, Abu-Pro- Ile, Aib-Pro-Ile, Aze-Pro-Ile, Cha-pro-Ile, Ile-Hyp-Ile, Ile-Pro- allo- Ile, Ile-Pro-t-Butyl-Gly, Ile-Pro-Val, Nle-Pro-Ile, Nva-Pro-Ile, Orn-Pro-Ile, Phe-Pro-Ile, Phg-Pro-Ile, Pip- Pro-Ile, Ser(Bzl)-Pro-Ile, Ser(P)-Pro-Ile, Ser-Pro-Ile, t-butyl-Gly-Pro-D-Val, t-butyl-Gly-Pro-Gly, t-butyl-Gly-Pro-Ile, t-butyl-Gly-Pro-Ile-amide, t-butyl-Gly-Pro-t-butyl-Gly, t-butyl-Gly-Pro-Val, Thr-Pro- Ile, Tic-Pro-Ile, Trp-Pro-Ile, Tyr(P)-Pro-Ile, Tyr-Pro- Allo -Ile, Val-Pro-Allo -Ile, Val-Pro-t-Butyl-Gly, Val -Pro-Val and pharmaceutically acceptable salts thereof, wherein t-butyl-Gly is defined by the formula

In addition, Ser(Bzl) and Ser(P) are defined as benzyl-serine and phosphoryl-serine, respectively. Tyr(P) is defined as phosphoryl-tyrosine. These compounds are disclosed in WO 03/002593.

More preferred DP IV-inhibitors that can be used in combination with a QC effector according to the present invention are peptidyl ketones, for example,

2-Methylcarbonyl-1-N-[(L)-alanyl-(L)-valinyl]-(2S)-pyrrolidine hydrobromide, 2-methylcarbonyl-1-N-[(L )-Valinyl-(L)-prolyl-(L)-valinyl]-(2S)-pyrrolidine hydrobromide,

2-[(acetyl-oxy-methyl)carbonyl]-1-N-[(L)-alanyl-(L)-valinyl]-(2S)-pyrrolidine hydrobromide,

2-[(benzoyl-oxy-methyl)carbonyl]-1-N-[{(L)-alanyl}-(L)-valinyl]-(2S)-pyrrolidine hydrobromide,

2-{[(2,6-dichlorobenzyl)thiomethyl]carbonyl}-1-N-[{(L)-alanyl}-(L)-valinyl]-(2S)-pyrrolidine,

2-[(benzoyl-oxy-methyl)carbonyl]-1-N-[glycyl-(L)-valinyl]-(2S)-pyrrolidine hydrobromide,

2-[([1,3]-thiazolethiazol-2-yl)carbonyl]-1-N-[{(L)-alanyl}-(L)-valinyl]-(2S) -Pyrrolidine trifluoroacetate,

2-[(benzothiazoethiazol-2-yl)carbonyl]-1-N-[N-{(L)-alanyl}-(L)-valinyl]-(2S)-pyrrolidine Trifluoroacetate,

2-[(benzothiazoethiazol-2-yl)carbonyl]-1-N-[{(L)-alanyl}-glycyl]-(2S)-pyrrolidine trifluoroacetide,

2-[(pyridin-2-yl)carbonyl]-1-N-[N-{(L)-alanyl}-(L)-valinyl]-(2S)-pyrrolidine trifluoroacetate

And other pharmaceutically acceptable salts thereof. These compounds are disclosed in WO 03/033524.

Furthermore, according to the present invention, substituted aminoketones can be used in combination with a QC effector. Preferred substituted amino ketones are,

1-cyclopentyl-3-methyl-1-oxo-2-pentanaminium chloride,

1-cyclopentyl-3-methyl-1-oxo-2-butanaminium chloride,

1-cyclopentyl-3,3-dimethyl-1-oxo-2-butanaminium chloride,

1-cyclohexyl-3,3-dimethyl-1-oxo-2-butanaminium chloride,

3-(cyclopentylcarbonyl)-1,2,3,4-tetrahydroisoquinolinium chloride,

N-(2-cyclopentyl-2-oxoethyl)cyclohexanaminium chloride and other pharmaceutically acceptable salts thereof.

Among the rare group of proline-specific proteases, DP IV was originally thought to be the only membrane-binding enzyme specific for proline as the second residue from the amino-terminus to the end of the polypeptide chain. However, other molecules, which are not structurally similar to DP IV, but even those with corresponding enzymatic activity were identified. DP IV-like enzymes identified to date include, for example, fibroblast activating protein α, dipeptidyl peptidase IV β, dipeptidyl aminopeptidase-like protein, N-acetylated α-linked acidic dipeptidase. , As quiescent cell proline dipeptidase, dipeptidyl peptidase II, attractin and dipeptidyl peptidase IV related protein (DPP 8), DPL1 (DPX, DP6), DPL2 and DPP 9 , Sedo & Malik (Sedo and Malik 2001, Biochim Biophys Acta, 36506,1-10) and Abbott and Gorrell (Abbott, CA and Gorrell, MD 2002 In: Langner & Ansorge (ed.), Ectopeptidases.Kluwer Academic/Plenum Publishers) , New York, 171-195). Recently, the cloning and characterization of dipeptidyl peptidase 10 (DPP 10) has been reported (Qi, S. Y. et al., Biochemical Journal Immediate Publication. Published on 28 Mar 2003 as manuscript BJ20021914).

Effectors, as used herein, are defined as molecules that increase their activity by binding to enzymes in vitro and/or in vivo. Some enzymes have binding sites for small molecules that affect their catalytic activity; The stimulating molecule is referred to as an activator. Enzymes can have multiple sites that recognize more than one activator or inhibitor. Enzymes can recognize the concentration of various molecules and use that information to alter their own activity.

Effectors can modulate enzyme activity because the enzyme can take both active and inactive forms: the activator is a positive effector, and the inhibitor is a negative effector. Effectors act not only at the active site of the enzyme, but also at the regulatory or allosteric site, the term stressing that the regulatory site is the part of the enzyme that is remote from the catalytic site, and at the catalytic site the regulation of this type is used to distinguish between the substrate and the competitive inhibitor and (Darnell, J., Lodish, H. and Baltimore, D. 1990, Molecular Cell Biology 2 nd Edition, Scientific American Books, New York, 63 if).

Preferred effectors according to the invention are inhibitors of QC- and EC-activity. Most preferred are competitive inhibitors of QC- and EC- activity.

Suitably, it is an activator of QC- and EC-activity.

In the peptides of the present invention, each amino acid residue is represented by a single-letter symbol or a three-letter symbol, as shown in the following list, corresponding to the common name of the amino acid:

As used herein, the term “QC” includes glutaminyl cyclase (QC) and QC-like enzymes. QC and QC-like enzymes are defined as the same or similar enzyme activity, further QC activity. In this regard, QC-like enzymes basically differ in molecular structure from QC.

As used herein, the term "QC activity" refers to the N-terminal glutamine residue as pyroglutamic acid (pGlu ^* ) under ammonia excretion, or the N-terminal L-homoglutamine or L-β-homoglutamine as cyclic pyro-homo. It is defined as intramolecular cyclization with glutamine derivatives (see Schemes 1 and 2).

[Scheme 1]

Cyclization of glutamine by QC

[Scheme 2]

Cyclization of L-homoglutamine by QC

As used herein, the term “EC” is defined as QC and the secondary activity of QC-like enzymes as glutamate cyclase (EC), further EC activity.

As used herein, the term "EC activity" is defined as the intramolecular cyclization of the N-terminal glutamate residue to pyroglutamic acid (pGlu ^{*) by QC (see Scheme 3).}

As used herein, the term "metal-dependent enzyme" refers to enzyme(s) that require bound metal ions to perform a catalytic function and/or bind metal ions to form a catalytically active structure. ).

[Scheme 3]

N-terminal cyclization of uncharged glutamyl peptide by QC (EC)

Another aspect of the present invention is to identify a novel physiological substrate of QC. These were identified by performing cyclization experiments using mammalian peptides, as described in Example 5. First, human QC and papaya QC were isolated as described in Example 1. The method applied is described in Example 2 and the peptide synthesis used is outlined in Example 6. Table 1 shows the results of the study.

New physiological substrate of glutaminyl cyclase temperament Human QC Papaya QC K _M
(μM) k _cat
(s ^-1 ) k _cat
(mM ^-1 s ^-1 ) K _M
(μM) k _cat
(s ^-1 ) k _cat /K _M
(mM ^-1 s ^-1 ) [Gln ¹ ]-gastrin 31±1 54.1±0.6 1745.2±36.9 34±2 25.8±0.5 759±30 [Gln ¹ ]-Nurotensin 37±1 48.8±0.4 1318.9±24.8 40±3 35.7±0.9 893±44 [Gln ¹ ]-FPP 87±2 69.6±0.3 800.0±14.9 232±9 32.5±0.4 140±4 [Gln ¹ ]-TRH 90±4 82.8±1.2 920.0±27.6 n.d n.d. n.d. [Gln ¹ ]-GnRH 53±3 69.2±1.1 1305.7±53.2 169±9 82.5±1.9 488.2±14.8 [Gln ³ ]-Glucagon (3-29) * * [Gln ⁵ ]-substrate P (5-11) * *

^* , Qualitatively determined by MALDI-TOF experiment

All assays were performed in the range of optimal activity and stability of human or plant QC shown in Example 4.

The amino acid sequence of a physiologically active peptide having a glutamine residue at the N-terminus and thus a substrate for the QC enzyme is listed in Table 2:

Amino acid sequence of a physiologically active peptide having a glutamine residue at the N-terminus that is converted to pyroglutamic acid (pGlu) after translation Peptide Amino acid sequence function Gastrin 17
Swiss-Prot:P01350 QGPWL EEEEEAYGWM DF (amide) Gastrin stimulates the gastric mucosa to produce and secrete hydrochloric acid, and it stimulates pancreas to secrete its digestive enzymes. It also stimulates smooth muscle contraction in the stomach and intestines, increases blood circulation and water secretion. Neurotensin
Swiss-Prot: P30990 QLYENKPRRP YIL Neurotensin plays an endocrine or paracrine role in regulating fat metabolism. Causes contraction of smooth muscles. FPP QEP Amide Tripeptides related to tyrotropin-releasing hormone (TRH) are found in the seminal plasma. Recently, there has been evidence that FPP plays an important role in regulating sperm fertility in vitro and in vivo. TRH
Swiss-Prot: P20396 QHP Amide TRH acts as a biosynthetic regulator of TSH in the anterior pituitary gland and as a neurotransmitter/neuromodulator of the central and peripheral nervous systems. GnRH
Swiss-Prot: P01148 QHWSYGL RP(G) amide Stimulate the secretion of gonadotropin; It stimulates the secretion of both luteinizing hormone and follicle-stimulating hormone. CCL16
(Small inducible cytokine A16)
Swiss-Prot:015467 QPKVPEW VNTPSTCCLK YYEKVLPRRL VVGYRKALNC
HLPAIIFVTK RNREVCTNPN
DDWVQEYIKD PNLPLLPTRN
LSTVKIITAK NGQPQLLNSQ It shows chemotactic activity on lymphocytes and monocytes, but not neutrophils. It also shows strong myelosuppressive activity and inhibits the proliferation of myeloblasts. Recombinant SCYA16 shows chemotactic activity against monocytes and THP-1 monocytes, but not resting lymphocytes and neutrophils. Pre-expression with RANTES to induce calcium flow in desensitized THP-1 cells. CCL8
(Small inducible cytokine A8)
Swiss-Prot: P80075 QPDSVSI PITCCFNVIN
RKIPIQRLES YTRITNIQCP
KEAVIFKTKR GKEVCADPKE
RWVRDSMKHL DQIFQNLKP A chemotactic agent that attracts monocytes, lymphocytes, basophils and eosinophils. Possibility to play a role in neoplasm formation and inflammatory host response. This protein can bind heparin. CCL2
(Small inducible cytokine A2)
Swiss-Prot: P13500 QPDAINA PVTCCYNFTN
RKISVQRLAS YRRITSSKCP
KEAVIFKTIV AKEICADPKQ
KWVQDSMDHL DKQTQTPKT Chemotactic agents that attract monocytes and basophils, but not neutrophils or eosinophils. Increases monocyte anti-tumor activity. Associated with the pathology of diseases characterized by mononuclear infiltration such as psoriasis, rheumatoid arthritis or atherosclerosis. Can play a role in dragging monocytes to the arterial wall during disease progression of atherosclerosis. Binds to CCR2 and CCR4. CCL18
Small inducible cytokine A18)
Swiss-Prot: P55774 QVGTNKELC CLVYTSWQIP
QKFIVDYSET SPQCPKPGVI
LLTKRGRQIC ADPNKKWVQK
YISDLKLNA A chemotactic agent that attracts lymphocytes, but not monocytes or granulocytes. Possibility of involvement in the migration of B cells from the lymph nodes to the B cell sac. In lymph nodes, it attracts intact T lymphocytes towards dendritic cells and activated macrophages, has chemotactic activity against native T cells, CD4+ and CD8+ T cells, and can thus play a role in humoral and cell-mediated immune responses. . Fractal Kin
(neurotactin)
Swiss-Prot: P78423 QHHGVT KCNITCSKMT
SKIPVALLIH YQQNQASCGK
RAIILETRQH RLFCADPKEQ
WVKDAMQHLD RQAAALTRNG
GTFEKQIGEV KPRTTPAAGG
MDESWLEPE ATGESSSLEP
TPSSQEAQRA LGTSPELPTG
VTGSSGTRLP PTPKAQDGGP
VGTELFRVPP VSTAATWQSS
APHQPGPSLW AEAKTSEAPS
TQDPSTQAST ASSPAPEENA
PSEGQRVWGQ GQSPRPENSL
EREEMGPVPA HTDAFQDWGP
GSMAHVSVVP VSSEGTPSRE
PVASGSWTPK AEEPIHATMD
PQRLGVLITP VPDAQAATRR
QAVGLLAFLG LLFCLGVAMF
TYQSLQGCPR KMAGEMAEGL
RYIPRSCGSN SYVLVPV The lytic form is chemotactic for T cells and monocytes, but not for neutrophils. The membrane-bound form promotes the adsorption of leukocytes to endothelial cells. Role in the process of leukocyte adsorption and migration in the endothelium. bind to cx3cr1 CCL7
(Small inducible cytokine A7)
Swiss-Prot: P80098 QPVGINT STTCCYRFIN
KKIPKQRLES YRRTTSSHCP
REAVIFKTKL DKEICADPTQ
KWVQDFMKHL DKKTQTPKL Chemotactic agents that attract monocytes and basophils, but neutrophils do not. Increases monocyte anti-tumor activity. It also induces the release of gelatinase B. This protein can bind to heparin. Binding to CCR1, CCR2 and CCR3. Orexin A
(Hypocretin-1)
Swiss-Prot O43612 QPLPDCCRQK TCSCRLYELL
HGAGNHAAGI LTL Neuropeptides that play an important role in regulating food intake and sleep-wake states, usually by harmonizing the complex behavioral and physiological responses of these complementary homeostasis functions. It also plays a role in energy metabolism, autonomic function, hormonal balance, and fluid regulation. Orexin-A binds both OX1 R and OX2R with high affinity. Temperament P RPK PQQFFGLM
(Cyclization of ^{Gln 5} after cleavage of residue 14) Belongs to the tachykinin class. Tachykinin is an active peptide that excites neurons and awakens behavioral responses, is a potent vasodilator and insulin secretagogue, and it contracts (directly or indirectly) many smooth muscles.

In the fourth embodiment, the peptide Gln ¹ - gastrin (17 and 34 amino acids in length), Gln ¹ - neurotensin and Gln ¹ -FPP was identified as new physiological substrates of QC. Gastrin, neurotensin and FPP contain a pGlu residue at their N-terminal position. It has been shown that these N-terminal pGlu residues are formed from N-terminal glutamine by QC catalysis for all peptides. As a result, when these peptides convert the N-terminal glutamine residue into pGlu, their biological function is activated.

Transepithelial transducing cells, especially gastrin (G) cells, coordinate the arrival of food in the stomach and secretion of gastric acid. Recent studies have shown that a number of active products are produced from gastrin precursors, and that there are a number of control points in gastrin biosynthesis. Biosynthetic precursors and intermediates (progastrin and Gly-gastrin) are putative growth factors; Their products, i.e. amideized gastrins, are genes involved in epithelial cell proliferation, differentiation of acid-producing body ganglion cells and histamine-secreting enterochromaffinity-like (ECL) cells, and histamine synthesis and storage in ECL cells. It regulates the expression of, as well as acutely stimulates acid secretion. Gastrin also stimulates the production of members of the epidermal growth factor (EGF) family, which in turn inhibits cellular function but stimulates the growth of superficial epithelial cells. Subjects infected with Helicobacter pylori, known to have an increased risk of duodenal ulcer disease and gastric cancer, have high plasma gastrin concentrations (Dockray, G. J. 1999 J Physiol 15 315-324).

The peptide hormone gastrin, which is released from antral G cells, is known to stimulate the synthesis and release of histamine from ECL cells through the CCK-2 receptor in the oxyntic mucosa. The transferred histamine induces acid secretion by binding to the H(2) receptor located on the cells of the body cavity wall. Recent studies also suggest that gastrin is a growth factor for the gastrointestinal system, both in its fully amideized and less treated forms (progastrin and glycine-extended gastrin). The main tropic effect of amidated gastrin is for the gastric acid-secreting mucosa, where it increases the proliferation of gastric stem cells and ECL cells, resulting in increased body cavity wall and ECL cell mass. It is established that it gives birth to. On the other hand, the main tropic effect of the less treated gastrin (eg glycine-extended gastrin) appears to be the colonic mucosa (Koh, T. J. and Chen, D. 2000 Regul Pept 9, 337-44).

In a fifth embodiment, the present invention uses the QC activity increasing effector to stimulate gastrointestinal cell proliferation, especially gastric mucosal cell proliferation, epithelial cell proliferation, and acid-producing body cavity wall cells and histamine-secreting enterochrome affinity-like (ECL ) To differentiate cells, express genes associated with histamine synthesis and storage in ECL cells, as well as to stimulate acute acid secretion in mammals by maintaining or increasing the concentration of ^{active [pGlu 1 ]gastrin.} to provide.

In a sixth embodiment, the present invention ^{reduces the conversion rate of inactive Gln 1} -gastrin to active [pGlu ¹ ]gastrin, thereby causing duodenal ulcer disease and gastric cancer, colon cancer, and sleepiness caused by or unrelated to Helicobacter pylori infection in mammals. Provides the use of an effector for reducing QC activity for the treatment of Zolliger-Ellison Syndrome.

Neurotensin (NT) is a neuropeptide involved in the pathophysiology of schizophrenia, and specifically regulates the neurotransmitter system previously shown to be misregulated in these diseases. Clinical studies measuring cerebrospinal fluid (CSF) NT concentrations have shown that reduced CSF NT concentrations in a series of schizophrenic patients are associated with effective antipsychotic medications. The behavioral and biochemical effects of NT administered spinally are remarkably similar to those of systemically administered antipsychotic drugs, and antipsychotic drugs increase NT neurotransmission. Consequently, NT functions as an endogenous antipsychotic agent. Moreover, typical and atypical antipsychotic drugs differently alter NT neurotransmission in the nigrastriatal and dopamine-terminal regions of the midbrain limbic system, and these effects can predict side effects disorders and efficacy, respectively (Binder, EB, etc. 2001 Biol Psychiatry 50, 856-872).

In a seventh embodiment, the present invention provides the use of an effector of increasing QC activity for the manufacture of an antipsychotic drug and/or for the treatment of schizophrenia in a mammal. The QC effector maintains or increases the concentration of active [pGlu ^{1] neurotensin.}

Fertilization prompting peptide (FPP), that is, tripeptide associated with thyrotrophin releasing hormone (TRH), is found in the intestine. Recent evidence obtained in vitro and in vivo has shown that FPP plays an important role in regulating sperm fertility. In particular, FPP stimulates non-fertile (non-reproductive) sperm to "switch on" and allows for faster fertilization, but spontaneous acrosome loss of sperm proceeds later. It prevents capacitation to prevent loss of reproductive potential. These reactions are mimicked and augmented by adenosine, known to regulate the adenylyl cyclase (AC)/cAMP signaling pathway. Both FPP and adenosine stimulate cAMP production in non-reproductive cells, but inhibit it in fertility cells, and interact with FPP receptors with adenosine receptors and G proteins to some extent to achieve the regulation of AC. These actions affect the tyrosine phosphorylation status of various proteins, some of which are important for the initial "initiation of action" and others related to the acrosome reaction itself. Calcitonin and angiotensin II are also found in the intestinal tract, have similar effects in vitro on non-reproductive sperm, and can increase the response to FPP. These molecules have similar effects in vivo and affect fertility by stimulating and maintaining fertility potential. Decreased availability of FPP, adenosine, calcitonin, and angiotensin II or defects in their receptors leads to male infertility (Fraser, L. R. and Adeoya-Osiguwa, S. A. 2001 Vitam Horm 63, 1-28).

In an eighth embodiment, the present invention provides the use of a QC activity-lowering effector for the manufacture of a contraceptive drug and/or for the manufacture of a drug that reduces the fertility of a mammal. QC activity lowering effectors ^{reduce the concentration of active [pGlu 1} ]FPP, preventing sperm fertility and leading to inactivation of sperm cells, whereas QC activity increasing effectors can stimulate male fertility and treat infertility. appear.

In a ninth embodiment, the physiological substrate of an additional QC is identified within the present invention. These are Gin ¹ -CCL2, Gin ¹ -CCL7, Gin ¹ -CCL8, Gin ¹ -CCL16, Gin ¹ -CCL18 and Gin ¹ -fractalkin (see Table 2 for details). These polypeptides include myeloid blast proliferation, neoplasm formation, inflammatory host response, cancer, psoriasis, rheumatoid arthritis, atherosclerosis, humoral and cellular-immune reactions, inhibition of leukocyte adsorption and migration processes in the endothelium, and Alzheimer's disease. It plays an important role in related inflammatory processes, pathophysiological conditions such as FBD and FDD.

Vaccines based on several cytotoxic T lymphocyte peptides against hepatitis B, human immunodeficiency virus and melanoma have been studied in recent clinical trials. One interesting melanoma vaccine candidate, alone or in combination with other tumor antigens, is the decapeptide ELA. These peptides are Melan-A/MART-1 antigen immunodominant peptide analogs with N-terminal glutamic acid. It has been reported that the amino group and gamma-carboxyl group of glutamic acid, as well as the amino group and gamma-carboxamide group of glutamine, are easily condensed to form pyroglutamic derivatives. To overcome this stability problem, several peptides of pharmaceutically interest have been developed with pyroglutamic acid instead of N-terminal glutamine or glutamic acid without loss of pharmaceutical properties. Unfortunately, compared to ELA, pyroglutamic acid derivatives (PyrELA) and N-terminal acetyl-capped derivatives (AcELA) failed to elicit cytotoxic T lymphocyte (CTL) activity. Despite the small changes on the surface introduced in PyrELA and AcELA, these two derivatives usually have a lower affinity than ELA for the specificity class I major histocompatibility complex. In conclusion, in order to preserve the overall activity of ELA, the formation of PyrELA should be avoided (Beck A. et al. 2001, J Pept Res 57, 528-38.). Recently, it was also discovered that the enzyme glutaminyl cyclase (QC) is overexpressed in melanoma (Ross D. T et al., 2000, Nat Genet 24, 227-35.).

In a tenth embodiment, the present invention relates to myeloid blast proliferation, neoplasm formation, inflammatory host response, cancer, malignant metastasis, melanoma, psoriasis, rheumatoid arthritis, atherosclerosis, humoral and cell-mediated immune response disorders, It provides the use of a QC effector for the manufacture of pharmaceuticals for the treatment of pathophysiological conditions such as inhibition of leukocyte adsorption and migration processes in endothelial tissues, and inflammatory processes associated with Alzheimer's disease, and pathophysiological conditions such as FBD and FDD.

In the eleventh embodiment, Gln ¹ -orexin A was identified as a physiological substrate of QC in the present invention. Orexin A is a neuropeptide that plays an important role in regulating food intake and sleep-wake state, usually harmonizing the complex behavioral and physiological responses of these complementary homeostasis functions. It also plays a role in energy metabolism, autonomic function, hormone balance and fluid regulation.

In the twelfth embodiment, the present invention provides a QC effector for the manufacture of a medicament for treating disorders in food intake and sleep-wake state, disorders in homeostasis of energy metabolism, disorders in autonomic nerve dysfunction, disorders in hormonal balance, and disorders in fluid control. Provides the use of.

The expansion of polyglutamine in several proteins leads to neurodegenerative diseases such as Parkinson's disease and Kennedy's disease. Therefore, the mechanism remains mostly unknown. The biochemical properties of the polyglutamine repeats suggest one possible explanation: pyroglutamate formation after internal cleavage at the glutaminyl-glutaminyl bond, the physicochemical stability, hydrophobicity, and amyloid formation of the polyglutamine protein. And it may contribute to the onset through an increase in neurotoxicity (Saido, T; Med Hypotheses (2000) Mar; 54, 427-9).

In a thirteenth embodiment, the present invention thus provides the use of a QC effector for the manufacture of a medicament for the treatment of Parkinson's disease and Huntington's disease.

In a fourteenth embodiment, the present invention provides a general method of reducing or inhibiting the enzymatic activity of QC. Examples of inhibitory compounds are also provided.

Inhibition of mammalian QC was initially detected only for 1,10-phenanthroline and reduced 6-methylpterin (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536). EDTA does not inhibit QC, thus leading to the conclusion that QC is not a metal-dependent enzyme (Busby, WHJ et al. 1987 J Biol Chem 262,8532-8536, Bateman, RCJ et al. 2001 Biochemistry 40, 11246-11250, Booth, RE et al. 2004 BMC Biology 2). However, in the present invention, the inhibitory properties of QC by 1,10-phenanthroline, dipicolinic acid, 8-hydroxy-quinoline and other chelators (FIGS. 18 and 19) and reactivation of QC by transition metal ions As revealed by (FIG. 20 ), it was found that human QCs and other animal QCs are metal-dependent enzymes. Finally, the metal dependence is shown by sequence comparison with other metal-dependent enzymes, showing that human QCs are also conserved with chelating amino acid residues (Fig. 21). The interaction of compounds with active-site binding metal ions presents a general method of reducing or inhibiting QC activity. In the present invention, it appears that imidazole derivatives are potent QC inhibitors. Using a serial assay (see Example 2 for details), many imidazole derivatives were analyzed for their ability to inhibit human QC as members of highly conserved mammalian QCs.

Accordingly, the present invention provides an imidazole derivative and histidine and derivatives thereof as an effector for reducing QC activity and properties thereof in terms of the type and potential of inhibition. The structure and K _i -values are shown in Tables 3 and 4. The results are described in detail in Example 7.

Inhibition constant of imidazole derivatives in human QC catalyzed reactions. Determination was carried out at 30° C. with 0.05 M Tris-HCl pH 8.0, including 5 mM EDTA. compound K _i -value (mM) rescue Central structure Imidazole 0.103±0.004 Benzimidazole 0.138±0.005 N-1 derivative 1-benzylimidazole 0.0071±0.0003 1-methylimidazole 0.030±0.001 1-vinylimidazole 0.049±0.002 Oxalic acid diimidazolidide 0.078±0.002 N-acetylimidazole 0.107±0.003 N-(trimethylsilyl)-imidazole 0.167±0.007 N-benzoylimidazole 0.174±0.007 1-(2-oxo-2-phenyl-ethyl)-imidazole 0.184±0.005 1-(3-aminopropyl)-imidazole 0.41±0.01 1-phenylimidazole No inhibition 1,1'-sulfonyldiimidazole No inhibition C-4(5) derivative N-omega-acetylhistamine 0.017±0.001 L-histidineamide 0.56±0.04 H-His-Trp-OH 0.60±0.03 L-histidineol 1.53±0.12 L-histidine 4.4±0.2 4-imidazole-carboxaldehyde 7.6±0.7 Imidazole-4-carboxylic acid 14.5±0.6 Methyl ester L-histamine 0.85±0.04 C-4,5 derivative 5-hydroxymethyl-4-methyl-imidazole 0.129±0.005 4-Amino-imidazole-5-carboxylic acid amide 15.5±0.5 4,5-diphenyl-imidazole No inhibition 4,5-dicyanoimidazole No inhibition C-2 derivative 2-methyl-benzylimidazole 0.165±0.004 2-ethyl-4-methyl-imidazole 0.58±0.04 2-aminobenzimidazole 1.8±0.1 2-chloro-1H-benzimidazole No inhibition Etc 3-(1H-imidazol-1-yl)-1-(3-methylbenzo[b]thiophen-2-yl)propan-1-one 0.0025±0.0001

4-[(1-methyl-1H-imidazol-5-yl)methyl]-3-propyldihydrofuran-2-(3H)-one 0.0067±0.0003

4-[2-(1H-imidazol-1-yl)-ethoxy]benzoic acid 0.0034±0.0001

3-[3-(1H-imidazol-1-yl)propyl]-2-thioxoimidazolidin-4-one 0.00081±0.00001

5-Nitro-2-[2-([{3-(1H-imidazol-1-yl-)propyl}amino]carbonyl)phenyl]furamide 0.0066±0.0004

N-(4-chlorophenyl)-N'-[2-(1H-imidazole-
1-yl)ethyl]thiourea 0.00165±0.00007

0.0322±0.0007

n.d. Imidazo<1.5a> pyridine 0.0356±0.0005

Methyl (2S)-2-{[(2S)-2-amino-5-(1H-imidazol-1-ylamino)-5-oxopentanoyl]amino}-3-methylbutanoate 0.164±0.004

QC inhibition by L-histamine and its two biological metabolites (also known as tele-methylhistamine) compound K _i value (mM) rescue L-histamine 0.85±0.04

3-Methyl-4-(β-aminoethyl)-imidazole 0.120±0.004

1-Methyl-4-(β-aminoethyl)-imidazole n.i.

In a fifteenth embodiment, novel new inhibitors of QC and QC-like enzymes based on cystiamine are provided.

In addition to imidazole derivatives and hydroxamates, thiol agents are often described as inhibitors of metal-dependent enzymes (Lowther, WT and Matthews, BW 2002 Chem Rev 102, 4581-4607; Lipscomb, W. N and Strater, N. 1996 Chem Rev 96, 2375-2433). In addition, thiol peptides have been described as inhibitors of QC associated with the aminopeptidase of Clan MH (Huntington, K. M. et. al. 1999 Biochemistry 38, 15587-15596). Although these inhibitors are inactive against mammalian QCs, they make it possible to isolate cystiamine derivatives as potent competitive QC-inhibitors (FIG. 23 ).

The present invention generally provides a QC-inhibitor or a pharmaceutically acceptable salt thereof, which may be represented by the following formula (1), and includes all stereoisomers:

Formula 1

In the above formula,

R ¹ -R ⁶ are each H or a branched or unbranched alkyl chain, a branched or unbranched alkenyl chain, a branched or unbranched alkynyl chain, carbocyclic, aryl, heteroaryl, heterocyclic , Aza-amino acids, amino acids or analogs thereof, peptides or analogs thereof; All of the above residues may be optionally substituted,

n is 0, 1 or 2, preferably 1, most preferably 0.

Throughout the specification and claims, "alkyl" can be represented by C _{1 -50} alkyl group, preferably C _{1 -30} alkyl groups, especially C _{1 -12} alkyl or C _{1 -8;} For example, the alkyl group can be a methyl, ethyl, propyl, isopropyl or butyl group. The expression "alk", for example the expression "alkoxy", and the expression "alkan", such as the expression "alkanoyl" can be defined as "alkyl"; The aromatic ("aryl") compound is preferably a substituted or optionally unsubstituted phenyl, benzyl, naphthyl, biphenyl or anthracene group, which preferably has at least 8C atoms; The expression "alkenyl" may be represented by a C _{2 -10} alkenyl group, preferably a C _{2 -6} alkenyl group, which may be substituted or unsubstituted with a double bond at any desired position; The expression "alkynyl" may be represented as a C _{2 -10} alkynyl group, preferably a C _{2 -6} alkynyl group, which may be substituted or unsubstituted with a triple bond at any desired position; The expression "substitured" or "substituent" may be any one or more, preferably one or two, alkyl, alkenyl, alkynyl, mono- or polyvalent acyl, alkanoyl, alkoxyalkanoyl or alkoxyalkyl groups. The desired substitution of may be indicated; Substituents mentioned above may have one or more (but preferably 0) alkyl, alkenyl, alkynyl, mono- or polyvalent acyl, alkanoyl, alkoxyalkanoyl or alkoxyalkyl groups in turn as side groups. I can.

The term "acyl" throughout the specification and the claims can be represented by C _{1 -20} acyl residue, preferably a C _{1 -8} acyl residue, particularly preferably a C _{1 -4} acyl residue; And "carbocyclic" may be represented by a C _{3 -12} carboxyl residue, preferably a C ₄ , C ₅ or C ₆ carboxyl residue. "Heteroaryl" may be defined as an aryl moiety, wherein 1 to 4, more preferably 1, 2 or 3 ring atoms are substituted with heteroatoms such as N, S or O. "Heterocyclic" is a cycloalkyl moiety, wherein 1, 2 or 3 ring atoms are substituted with heteroatoms such as N, S or O.

"Peptide mimetics" themselves are known to those of skill in the art. They are preferably defined as compositions having a secondary structure, such as a peptide, and optionally a composition having additional structured properties; Its mode of action is largely similar or identical to that of the original peptide; However, its action (eg, as an antagonist or inhibitor) can be altered for comparison with the original peptide, particularly a receptor or enzyme. Moreover, they can mimic the effects of native peptides (antagonists). Examples of peptide analogs are scaffold mimetics, non-peptidic analogs, peptoids, peptide nucleic acids, oligopyrrolinones, vinyllog peptides and oligocarbamates. Definitions of these peptide analogs can be found in Lexikon der Chemie, Spektrum Akademischer Verlag Heidelberg, Berlin, 1999.

"Aza-amino acid" is defined as an amino acid in which a chiral α-CH group is substituted with a nitrogen atom, whereas "aza-peptide" is a chiral α of one or more amino acid residues in the peptide chain. Refers to a peptide in which the -CH group is substituted with a nitrogen atom.

The roles of thiol and amino groups are shown by comparison of the inhibitory efficacy of dimethylcysteamine, cysteamine, mercaptoethanol, ethylenediamine, and ethanolamine, as shown in Table 5. Only compounds having amino and thiol groups are efficacious, and deletion or alteration of either group reduces the inhibitory ability. Moreover, the pH-dependence of the inhibition of murine QC by cysteamine, dimethylcysteamine and mercaptoethanol shows a difference, and the protonation state of the inhibition affects the binding of the inhibitor to the active site (Fig.22). ).

Comparison of the efficacy of cysteamine-derived compounds to inhibit QC (ni, concentration of 5 mM, pH = 8.0, 30° C. and _{no inhibition detected at 1K M} substrate concentration in the sample, nd, not measured) compound K _i -value( mM ) rescue Cysteamine 0.043±0.002

Dimethyl-cysteamine 0.0190±0.0002

Diethyl-cysteamine 0.0109±0.0004

Mercaptoethanol 3.91±0.08

Ethyl mercaptan n.d.

Ethylenediamine n.i.

Ethanolamine n.i.

Surprisingly, during characterization of enzymatic activity, in addition to the N-terminal glutaminyl residue, it was found that the N-terminal β-homo-glutaminyl residue also performs characterization as a QC substrate derived from plants and animals. The N-terminal β-homo-glutaminyl residue was converted into a five-membered lactam ring by catalysis of human and papaya QC, respectively. The results are described in Example 5. The applied method is shown in Example 2, and the peptide synthesis was carried out as described in Example 6.

Another preferred embodiment of the present invention includes a method of screening for QC effectors.

A preferred screening method for identifying QC activity altering effectors from a group of compounds comprises the following steps:

a) contacting the compounds and QC under conditions that permit bonding between them;

b) adding a QC substrate;

c) observing the conversion of the substrate or, optionally, measuring residual QC activity;

d) Calculating the change in the enzymatic activity of substrate conversion and/or QC to identify the effector of altering the activity.

Another preferred screening method relates to a method for the identification and selection of effectors that directly or indirectly interact with QC activity-site binding ions, comprising the following steps:

a) contacting the compound and QC under conditions that allow bonding between them;

b) adding a QC substrate that is converted by QC;

c) observing the conversion of the substrate or, optionally, measuring residual QC activity; And

d) calculating a change in substrate conversion and/or enzymatic activity of QC, wherein the change can be used to identify an effector that alters QC activity.

Preferred in the above-described screening method is mammalian QC or papaya QC. Particularly preferred is mammalian QC, since the effectors identified by this screening method will be used to treat diseases in mammals, especially humans.

The agent selected by the above-described screening method can work by lowering the conversion of at least one QC substrate (negative effector, inhibitor) or increasing the conversion of at least one QC substrate (positive effector, activator).

The compounds of the present invention can be converted into acid addition salts, in particular pharmaceutically acceptable acid addition salts.

The salt of the compound of the present invention may be in the form of an inorganic salt or an organic salt.

The compounds of the present invention can be converted and used into acid addition salts, in particular pharmaceutically acceptable acid addition salts. Pharmaceutically acceptable salts generally take a form in which the basic side chain is protonated with an inorganic or organic acid. Representative organic or inorganic acids are hydrochloric acid, hydrobromic acid, perchloric acid, sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, hydride. Oxyethanesulfonic acid, benzenesulfonic acid, oxalic acid, pamoic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, cyclohexanesulfonic acid, silicylic acid, sacharinic acid or trifluoroacetic acid. All pharmaceutically acceptable acid addition salt forms of the compounds of the present invention are included within the scope of the present invention.

In view of the close relationship between the free compounds and the compounds in the form of their salts, whenever a compound is referred to in the specification, the corresponding salt is intended to be included as well, if possible or appropriate under the conditions.

Thus, when the compounds according to the invention have at least one chiral center, they can exist as enantiomers. When the compounds have more than one chiral center, they may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are included within the scope of the present invention. Furthermore, some of the crystalline forms of the compounds may exist as polymorphs, which are intended to be included in the present invention. In addition, some of the compounds may form solvates (ie, hydrates) or conventional organic solvents in water, and such solvates are intended to be included within the scope of the present invention.

These compounds, including salts, may also be obtained in the form of their hydrates, or may contain other solvents used for their crystallization.

In a further embodiment, the present invention provides a method of preventing or treating a condition mediated by the modulation of QC enzyme activity in a subject in need thereof, which provides a large amount of any compound of the present invention or a pharmaceutical composition thereof. And administering as a therapeutically effective regimen for treating the condition. In addition, the present invention encompasses the use of the compounds of the present invention, and their corresponding pharmaceutically acceptable acid addition salt forms, for the manufacture of pharmaceuticals for preventing or treating conditions mediated by modulation of QC activity in a subject. do. The compounds can be administered to a patient by any conventional route of administration, including, but not limited to, intravenous, oral, subcutaneous, intramuscular, intradermal, parenteral, and combinations thereof.

In a further preferred embodiment, the invention relates to a pharmaceutical composition, i.e., a medicament comprising at least one compound of the invention or a salt thereof, optionally in combination with one or more pharmaceutically acceptable carriers and/or solvents. About.

The pharmaceutical composition may be, for example, in the form of a parenteral or enteral formulation and may include an appropriate carrier, or may be in an oral formulation including a carrier suitable for oral administration. Preferably, it is in the form of oral administration.

The QC activity effector administered according to the present invention may be a pharmaceutically administrable formulation or formulation complex as a QC- and EC- activity inhibitor, preferably a competitive inhibitor, or an enzyme inhibitor that lowers the QC protein concentration in a mammal, a competitive It can be used in combination with an enzyme inhibitor, a substrate, a pseudosubstrate, an inhibitor of QC expression, a binding protein, or an antibody of these enzyme proteins. The compounds of the present invention can be individually tailored to treatment for patients and diseases, especially to avoid individual tolerance, allergies and side effects.

These compounds also exhibit varying degrees of activity as a function of time. The treating physician is thus given the opportunity to react differently depending on the individual situation of the patient: he is able to precisely adjust the onset rate of action on the one hand, and the duration of action, in particular the intensity of action, on the other hand.

The preferred method of treatment according to the present invention presents a new approach to the prevention or treatment of conditions mediated by the regulation of QC enzyme activity in mammals. Advantageously, it is simple, commercially applicable and suitable for use in mammals, especially in human pharmaceuticals, in particular based on the disproportionate concentration of physiologically active QC substrates listed in, for example, Tables 1 and 2. It is suitable for use in the treatment of diseases.

For example, it is possible to advantageously administer the compound in the form of a pharmaceutical preparation comprising the combination of the active ingredient with conventional excipients such as diluents, excipients and/or carriers known from the prior art. For example, they can be administered parenterally (eg, contained in physiological saline and intravenously) or enterally (eg, formulated with conventional carriers, orally).

According to endogenous stability and bioavailability, one or more daily doses of the compounds may be presented to achieve a desired normoglycemic level. For example, this dosage range in the human body may range from about 0.01 mg to 250.0 mg per day, and preferably from about 0.01 to 100 mg of compound per kilogram of body weight.

By administering a QC activating effector to a mammal, Alzheimer's disease, Down's syndrome, family history British dementia (FBD) and family history Danish dementia (FDD), ulcer disease caused by or unrelated to Helicobacter pylori infection and gastric cancer, colorectal cancer, Jolliger- Ellison syndrome, pathogenic psychosis, schizophrenia, infertility, neoplasm formation, inflammatory host response, cancer, psoriasis, rheumatoid arthritis, atherosclerosis, humoral and cell-mediated immune response disorders, leukocyte adsorption and migration processes in the endothelium , Food intake disorders, sleep-wake conditions, disorders of homeostasis of energy metabolism, disorders of autonomic dysfunction, disorders of hormonal balance and disorders of fluid control.

Furthermore, by administering a QC activating effector to a mammal, gastrointestinal cell proliferation, preferably proliferation of gastric mucosa cells, epithelial cells, acute acid secretion of acid-producing body ganglion cells and histamine-secreting enterochrome affinity-like cells And it would be possible to stimulate differentiation.

In addition, administration of QC inhibitors to mammals leads to loss of sperm cell function, thus inhibiting male fertility. Accordingly, the present invention provides a method for regulating and regulating male fertility and a use for preparing an effector for reducing QC activity as a male contraceptive.

Furthermore, it may be possible to inhibit the proliferation of myeloid blasts by administering a QC activating effector to a mammal.

The compounds used according to the invention are therefore, in a manner originally known, using inert, non-toxic, pharmaceutically suitable carriers and excipients or solvents, for example tablets, capsules, sugars, pills, suppositories, granules. , Aerosols, syrups, liquid, solid and cream-like emulsions and suspensions and solutions. In each of these formulations, the therapeutically effective compound is preferably about 0.1 to 80% by weight, more preferably 1 to 50% by weight of the total mixture, ie in an amount sufficient for the aforementioned dosage level to be obtained.

These substrates can be used as drugs in the form of sugars, capsules, double capsules, tablets, drops, syrups, or also as suppositories or as nasal sprays.

Such formulations can advantageously be prepared, for example, by extending the active ingredient with a solvent and/or carrier, if possible, optionally with an emulsifier and/or dispersant aid, if for example water is used as a diluent. It can be used for an organic solvent that is selectively used as a solvent.

Examples of excipients useful in connection with the present invention include: water; Paraffins (e.g. natural oil), vegetable oils (e.g. rapeseed oil, ground fruit oil, sesame oil), alcohols (e.g. ethyl alcohol, glycerol), glycols (e.g. propylene glycol, polyethylene glycol) ) Non-toxic organic solvents such as; Solid carriers such as, for example, natural powdered minerals (eg highly dispersed silica, silicates), sugars (eg raw sugar, lactose and dextrose); Emulsifiers such as nonionic and anionic emulsifiers (for example, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, alkylsulfonates and arylsulfonates); Dispersants (eg lignin, sulfite liquid, methylcellulose, starch and polyvinylpyrrolidone); Lubricants (eg magnesium stearate, talcum, stearic acid and sodium lauryl sulfate) and optionally flavoring agents.

Administration can be carried out in a general manner, preferably enterally or parenterally, in particular orally. For enteral administration, tablets may contain, in addition to the aforementioned carriers, additional excipients such as sodium citrate, calcium carbonate and calcium phosphate, preferably with various excipients such as potato starch, gelatin, and the like. Furthermore, lubricants such as magnesium stearate, sodium lauryl sulfate and talcum can be used in combination with tabletting. In the case of aqueous suspensions and/or elcxirs for oral administration, neutralizing agents or coloring agents of various flavors may be added to the active ingredient in addition to the aforementioned excipients.

For parenteral administration, a solution of the active ingredient using a suitable liquid carrier can be used. In general, in the case of intravenous administration, in order to obtain an effective result, an amount of about 0.01 to 2.0 mg, preferably about 0.01 to 1.0 mg per kg of body weight per day is administered, and in the case of enteral administration, the dosage is per kg body weight. It is advantageous if it is about 0.01 to 2 mg per day, preferably about 0.01 to 1 mg per day.

However, in some cases, different dosages than those described above may be required depending on the body weight of the experimental animal or patient or the type of route of administration, as well as on the basis of the type of animal and the individual response to the drug, or the interval at which they are administered. Thus, in some cases it may be sufficient to use an amount less than the aforementioned minimum amount, while in other cases the aforementioned upper limit must be exceeded. When relatively large amounts are administered, it is desirable to divide these amounts into several single doses throughout the day. When administered as a human drug, the same dosage range as above is given. In this case, the above is similarly applied.

These and other aspects of the invention will be better understood by reference to the following figures:
1 shows the progress curve for cyclization of H-Gln-Ala-OH, catalyzed by human QC, by measuring the decrease in absorbance at 340 nm. Samples included 0.3 mM NADH/H ⁺ , 14 mM α-ketoglutaric acid, 30 U/ml glutam dehydrogenase and 1 mM H-Gln-Ala-OH. For curve AD, various concentrations of QC were applied: A, 10 mU/m, B, 5 mU/ml, C, 2.5 mU/ml. For curve D, QC was not applied. A linear association was observed between the QC concentration and the observed activity (degree of inset).
Figure 2 shows the pH dependence of human and papaya (inset) QCs as measured under the first order rate using Gln-βNA as a substrate. For human QC, a buffer system that provides constant ionic strength according to Ellis and Morrison, consisting of 25 mM MES, 25 mM acetic acid and 50 mM Tris (Ellis, KJ and Morrison, JF 1982 Methods Enzymol. 87,405-426) Was used. Due to the weak inhibitory effect of Tris, papaya QC was investigated using 50 mM Mops buffer. NaCl was added to adjust the ionic strength to 0.05M. The rate profile was evaluated by fitting a separator based model. _{For Papaya QC, pK a} 7.13 ±0.03 was obtained by fitting the data to a single separation model.
3 shows the effect of pH on the stability of QC and human QC derived from papaya latex. The enzyme stock solution was diluted 20-fold in 0.1 M buffer (pH 4-7 sodium citrate, pH 7-10 sodium phosphate) at various pH values. The enzyme solution was treated at 30° C. for 30 minutes and subsequently the enzyme activity was analyzed according to standard methods.
4 is a diagram comparing _{the specificity constant k cat} /K _M for a group of substrates including glutamate at the second amino acid position. While the specificity of human QC was increased from dipeptide or tetrapeptide, no change was observed for papaya QC. The data given here is a re-presentation of the parameters presented in Table 3.
Figure 5 shows the formation of pGlu-Lys(pGlu)-Arg-Leu-Ala-NH ₂ from H-Gln-Lys(Gln)-Arg-Leu-Ala-NH _{2 catalyzed by human QC.} Substrate conversion was observed as a time-dependent change in the m/z ratio due to the release of ammonia. The sample composition was 0.5 mM substrate, 40 mM Tris/HCl with 38 nM QC, pH 7.7. At the indicated times, the sample was removed from the analysis tube, mixed with the matrix solution (1:1 v/v), and the mass spectrum was recorded. Very similar dependence was observed for Papaya QC.
Figure 6 shows the formation of pGlu-Phe-Lys-Ala-Glu-NH ₂ from H-Gln(NMe)-Phe-Lys-Ala-Glu-NH _{2 catalyzed by Papaya QC.} Substrate conversion was observed as a time dependent change in the m/z ratio due to the release of methylamine. The sample composition was 0.5 mM substrate, 40 mM Tris/HCl containing 0.65 μM Papaya QC, pH 7.7. At the indicated times, the sample was removed from the analysis tube, mixed with the matrix solution (1:1 v/v), and the mass spectrum was recorded. No substrate conversion was observed in the sample without papaya QC or until human QC was applied to the substrate up to 1.5 μM (not shown).
Figure 7 shows the formation of Gln ³ -Aβ (3-11) from the, Gln ³ -Aβ (1-11) catalyzed by the DP Ⅳ. At the indicated times, the sample was removed from the analysis tube, mixed with the matrix solution (1:1 v/v), and the mass spectrum was recorded.
8 shows the prevention of cleavage of ^{Gln 3} -Aβ(1-11) by DP IV-inhibitor Val-pyrrolidide (Val-Pyrr). At the indicated times, the sample was removed from the analysis tube, mixed with the matrix solution (1:1 v/v), and the mass spectrum was recorded.
9 shows the production of ^{pGlu 3 -Aβ(3-11) from Gln 3} -Aβ(3-11), catalyzed by QC. At the indicated times, the sample was removed from the analysis tube, mixed with the matrix solution (1:1 v/v), and the mass spectrum was recorded.
^{10 shows the inhibition of [pGlu 3} ]Aβ(3-11) formation ^{from Gln 3} -Aβ(3-11) by the QC-inhibitor 1,10-phenanthroline. At the indicated times, the sample was removed from the analysis tube, mixed with the matrix solution (1:1 v/v), and the mass spectrum was recorded.
^{11 shows the formation of [pGlu 3} ]Aβ(3-11) ^{from Gln 3} -Aβ(1-11) after continuous catalysis by DP IV and QC. At the indicated times, the sample was removed from the analysis tube, mixed with the matrix solution (1:1 v/v), and the mass spectrum was recorded.
12 shows inhibition of the formation of ^{[pGlu 3 ]Aβ(3-11) from Gln 3} -Aβ(1-11) by QC-inhibitor 1,10-phenanthroline in the presence of catalytically active DP IV and QC Represents. At the indicated times, the sample was removed from the analysis tube, mixed with the matrix solution (1:1 v/v), and the mass spectrum was recorded.
13 shows the reduction in the formation of ^{[pGlu 3 ]Aβ(3-11) from Gln 3} -Aβ(1-11) by DP IV-inhibitor Val-Pyrr in the presence of catalytically active DP IV and QC. At the indicated times, the sample was removed from the analysis tube, mixed with the matrix solution (1:1 v/v), and the mass spectrum was recorded.
^{14 shows the formation of [pGlu 3} ]Aβ(3-11) ^{from Gln 3} -Aβ(1-11) after successive catalysis by aminopeptidase(s) and QC present in porcine pituitary gland . At the indicated times, the sample was removed from the analysis tube, mixed with the matrix solution (1:1 v/v), and the mass spectrum was recorded.
15A and 15B show mass spectra of Aβ(3-ll)a and Aβ(3-21)a incubated with recombinant human QC heated for 10 minutes before use. 15C and 15D are mass spectra of Aβ(3-11)a and Aβ(3-21)a in the presence of active human QC, respectively, [pGlu ³ ]Aβ(3-11)a and [pGlu ³ ]Aβ( 3-21) represents the formation of a. 15E and 15F are mass spectra of Aβ(3-11)a and Aβ(3-21)a in the presence of active QC and 5 mM benzimidazole, showing inhibition of ^{[pGlu 3] formation.}
Figure 16 shows the kinetics of papaya QC-catalyzed Glu-βNA-conversion plotted against substrate concentration. The onset rate was measured in 0.1 M pyrophosphate buffer, pH 6.1 (square), 0.1 M phosphate buffer, pH 7.5 (round) and 0.1 M borate buffer, pH 8.5 (triangle). The mechanical parameters are as follows: K _M = 1.13 ±0.07 mM, k _cat = 1.13 ±0.04 min ^-1 (pH 6.1); K _M = 1.45 ±0.03 mM, k _cat = 0.92 ±0.01 min ^-1 (pH 7.5); K _M = 1.76 ±0.06 mM, k _cat = 0.56 ±0.01 min ^-1 (pH 8.5).
Fig. 17 shows the pH-dependence of conversion of Gln-βNA (circular) and Glu-βNA (square), determined under first order rate law conditions (S<<K _{M ).} Substrate concentrations were 0.01 mM and 0.25 mM, respectively. For both crystals, a three-component buffer system consisting of 0.05 M acetic acid, 0.05 M pyrophosphate and 0.05 M trisine was applied. All buffers were adjusted to have the same conductivity by adding NaCl to avoid a difference in ionic strength. The data were fitted to the equations established for the two dissociating groups, and the pK _a -value was 6.91 ±0.02 for Gln-βNA, and 4.6 ±0.1 and 7.55 ±0.02 for 9.5 ±0.1 and Glu-βNA. Revealed. _{PK a} of each substrate amino group determined by titration Values were 6.97±0.01 (Gln-βNA) and 7.57±0.05 (Glu-βNA). All crystals were carried out at 30 °C.
18 shows the progress curve of cyclization of human QC-catalyzed H-Gln-AMC in the presence of imidazole, dipicolinic acid and in the absence of inhibitory compounds. The hyperbolic shape of the curve in the presence of dipicolinic acid indicates the removal of metal ions from the QC active site.
Figure 19 shows the time-dependent inactivation of QC by heterocyclic chelator 1,10-phenanthroline. After treatment of the QC-enzyme with an inhibitor in the absence of a substrate (continuous line), a reduced enzyme activity was observed compared to the sample not previously treated with the inhibitor (dotted line), indicating that metal ions were removed from the QC active site.
20 shows the reactivation of human QCs with monovalent- and divalent metal ions. QC was inactivated by adding 50 mM Bis-Tris containing 2 mM dipicolinic acid, pH 6.8. The enzyme was then dialyzed against 50 mM Bis-Tris, pH 6.8 containing 1.0 mM EDTA. For the reactivation of the enzyme, in order to avoid uncharacteristic reactivation by trace amounts of metal ions present in the buffer solution, the inactivated enzyme samples in the presence of 0.5 mM EDTA were treated with 0.5 mM metal ions. The enzyme sample dialyzed against the EDTA solution as non-inactivated, but inactivated enzyme (+EDTA) and the enzyme sample dialyzed against the buffer solution without the addition of EDTA (-EDTA) were used as controls.
Fig. 21 shows the arrangement of the sequences of human QC (hQC) and other M28 family members of the metalpeptidase Clan MH. Multiple sequence arrangements were performed using ClustalW from ch.EMBnet.org as setpoint. The preservation of zinc-ion ligation residues is human QC (hQC; GenBank X71125), Zn-dependent aminopeptidase from Streptomyces griseus (SGAP; Swiss-Prot P80561), and human It was found in the N-acetylated-alpha-linked acidic dipeptidase (NAALADase I) domain (residues 274-587) of glutamate carboxypeptidase II (hGCP II; Swiss-Prot Q04609). Amino acids involved in metal bonds are printed in bold and underlined. For human QC, these residues are putative counterparts to peptidases.
Fig. 22 shows that the inhibitor of murine QC shows pH-dependence by cysteamine (square), dimethyl-cysteamine (circle), and mercaptoethanol (triangle). The points were fitted to the formula set for one dissociation period. The curves appear in different shapes, indicating that the dependence is due to the change in the protonation of the inhibitor. The kinematically determined pK _a -value (8.07±0.07) for cysteamine and the value of dimethyl-cysteamine (8.07±0.03) are in good agreement with those obtained from literature values for amino groups (8.6 and 7.95, respectively) (Buist , GJ and Lucas, HJ 1957 J Am Chem Soc 79, 6157; Edsall, JT Biophysical Chemistry, Academic Press, Inc., New York, 1958). Thus, the pH-dependence of mercaptoethanol has a consistent slope, since it does not carry a dissociation phase in the investigated pH-category.
Figure 23 shows a double inverse plot (Lineweaver-Burk plot) obtained by conversion of Gln-AMC (0.25, 0.125, 0.063, 0.031 mM), the presence of various concentrations of cysteamine (0, 0.25, 0.5, 1 mM) Catalyzed by human QC. The data were fit according to competitive inhibitors. The determined Ki-value is 0.037±0.001 mM.
Peptide sequence
The peptides mentioned and used in the present invention have the following sequence:
Aβ(1-42) :
*Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe- Phe-Ala-Glu-Asp-Val-Gly -Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala
Aβ(1-40) :
Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe- Phe-Ala-Glu-Asp-Val-Gly- Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val
Aβ(3-42) :
Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn- Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-Ile-Ala
Aβ(3-40) :
Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala- Glu-Asp-Val-Gly-Ser-Asn- Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val
Aβ(1-11)a :
Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-NH ₂
Aβ(3-11)a :
Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-NH ₂
Aβ(1-21)a :
Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-NH ₂
Aβ(3-21)a :
Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-NH ₂
Gin ³ -Aβ(3-40) :
Gln-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn- Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val
Gln ³ -Aβ(3-21)a :
Gln-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-NH ₂
Gln ³ -Aβ(1-11)a :
Asp-Ala-Gln-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-NH ₂
Gln ³ -Aβ(3-11)a :
Gln-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-NH ₂
ABri
pGlu-Ala-Ser-Asn-Cys-Phe-Ala-Ile-Arg-His-Phe-Glu-Asn-Lys-Phe-Ala-Val-Glu- Thr-Leu-Ile-Cys-Ser-Arg-Thr- Val-Lys-Lys-Asn-Ile-Ile-Glu-Glu-Asn
Gln ¹ - ABri
Glu-Ala-Ser-Asn-Cys-Phe-Ala-Ile-Arg-His-Phe-Glu-Asn-Lys-Phe-Ala-Val-Glu-Thr-Leu-Ile-Cys-Ser-Arg-Thr- Val-Lys-Lys-Asn-Ile-Ile-Glu-Glu-Asn
ADan
pGlu-Ala-Ser-Asn-Cys-Phe-Ala-Ile-Arg-His-Phe-Glu-Asn-Lys-Phe-Ala-Val-Glu-Thr-Leu-Ile-Cys-Phe-Asn-Leu- Phe-Leu-Asn-Ser-Gln-Glu-Lys-His-Tyr
Glu ¹ - ADan
Glu-Ala-Ser-Asn-Cys-Phe-Ala-Ile-Arg-His-Phe-Glu-Asn-Lys-Phe-Ala-Val-Glu-Thr-Leu-Ile-Cys-Phe-Asn-Leu- Phe-Leu-Asn-Ser-Gln-Glu-Lys-His-Tyr

Pharmaceutical formulation Example

1. Capsules containing 100 mg each of the compounds of the present invention:

For about 10,000 capsules, a solution of the following composition was prepared:

1.0 kg of the compound of the present invention

0.5 kg of glycerol

3.0 kg of polyethylene glycol

0.5 kg of water

5.0 kg

The solution was put into a soft gelatin capsule by a known method. The capsules are suitable for chewing or flipping.

2. Tablets or coated tablets containing 100 mg of the compound of the present invention, or sugar:

100,000 tablets were prepared in the following amounts:

Compounds of the present invention,

Fine powder 10.0 kg

4.35 kg of glucose

4.35 kg lactose

4.50 kg of starch

Cellulose, fine powder 4.50 kg

After mixing the above ingredients, a solution prepared from the following was added.

2.0 kg of polyvinylpyrrolidone

*0.1 kg of polysorbate

Potion. 5.0 kg

Then, the wet mass was ground, 0.2 kg of magnesium stearate was added, and then granulated by a known method of drying. 30.0 kg of the finished tablet mixture was processed to form convex tablets weighing 300 mg. Ideally, the tablets were coated or sugar-coated in a known manner.

The pharmaceutical composition according to the invention advantageously comprises a combination of at least one effector of QC activity and at least one DP IV inhibitor. Such pharmaceutical compositions are particularly useful for the treatment of Alzheimer's disease and Down's syndrome.

Example 1: human and papaya QC Manufacture of

Host strain ( strain ) And badge

Pichia pastoris (P. pastoris, Pichia pastoris) strain x 33 ( AOX1 , AOX2 ) used for the expression of human QC was cultured, transformed and analyzed according to the manufacturer's instructions (Invitrogen). The media required for P. pastoris, namely buffered glycerol (BMGY) complex or methanol (BMMY) complex media, and fermentation basal salt media were prepared according to the manufacturer's recommendations.

human QC The molecule of the plasmid vector encoding Cloning

All cloning procedures were performed using standard molecular biology techniques. The vector pPICZαB (Invitrogen) was used for expression in yeast. Human QC was expressed in E. coli using the pQE-31 vector (Qiagen). The cDNA of mature QC starting with codon 38 was fused in frame with a plasmid encoding a 6xhistidine tag. After amplification and subcloning using the primers pQCyc-1 and pQCyc-2, a fragment was inserted into the expression vector using the restriction sites of SphI and HindIII.

P. Pastoris Transformation and small scale expression

Plasmid DNA was amplified in E. coli JM109 and purified according to the manufacturer's recommendation (Qiagen). In the expression plasmid pPICZαB used, three restriction sites were provided for linearization. Since SacI and BstXI are cleaved in QC cDNA, PmeI was selected. 20-30 μg plasmid DNA was linearized with PmeI, precipitated with ethanol, and dissolved in sterile deionized water. Then, 10 μg of the DNA was applied to the transformation of competent P. pastoris cells by electroporation according to the manufacturer's (BioRad) instructions. Selection was made on a plate containing 150 μg/ml Zeocin. Hundreds of transformants were obtained by transforming once using the linearized plasmid.

To test the recombinant yeast clone for QC expression, the recombinant was incubated for 24 hours in a 10 ml conical tube containing 2 ml BMGY. Then, the yeast was centrifuged and resuspended in 2 ml BMMY containing 0.5% methanol. This concentration was maintained by adding methanol every 24 hours until 72 hours. Then, the QC activity was determined in the supernatant. The presence of the fusion protein was confirmed by Western blot analysis using an antibody direct against the 6x histidine tag (Qiagen). Clones showing the highest QC activity were selected and used for further experiments and fermentation.

Large-scale expression in fermenters

Expression of QC was carried out in a 5L reactor (Biostat B, B. Braun biotech), in particular as described in the "Pichia Fermentation Process Guidelines" (Invitrogen). Briefly, cells were cultured in fermentation basal salt medium (pH 5.5) supplemented with trace salt and glycerol as the sole carbon source. During the first batch phase for about 24 hours and the fed-batch phase for the subsequent about 5 hours, cell mass accumulates. When the wet weight reached 200 g/l, QC expression was induced using methanol while applying a three-stage feeding process for a total of about 60 hours of fermentation. Subsequently, cells were removed from the QC-containing supernatant by centrifugation for 15 minutes at 4° C. at 6000×g speed. NaOH was added to adjust the pH to 6.8, and the resulting cloudy solution was centrifuged again at 4° C. for 40 minutes at a rate of 37000×g. If still turbid, an additional filtration step was applied using a cellulose membrane (pore width 0.45 μm).

P. In Pastoris Expressed 6x histidine label QC Tablets of

His-labeled QC was first purified by immobilized metal affinity chromatography (IMAC). ^{As a general purification, 1000 ml of culture supernatant was added to a Ni 2 +} -dropped Chelating Sepharose FF column (1.6 x 20 cm, Pharmacia) equilibrated with 750 mM NaCl, 50 mM phosphate buffer, and pH 6.8, at a flow rate of 5 ml/min. It was applied at a rate. After washing with 10 column volumes of equilibration buffer and 5 column volumes of stabilization buffer containing 5 mM histidine, the bound protein was eluted with 50 mM phosphate buffer, pH 6.8 containing 150 mM NaCl and 100 mM histidine. The obtained eluate was dialyzed against 20 mM Bis-Tris/HCl, pH 6.8 at 4° C. overnight. Subsequently, the QC was further purified by anion exchange chromatography on a Mono Q6 column (BioRad) equilibrated with dialysis buffer. The QC-containing fraction was loaded onto the column at a flow rate of 4 ml/min. The column was then washed with an equilibration buffer containing 100 mM NaCl. Elution was performed with two gradients representing equilibration buffers containing 240 mM and 360 mM NaCl at 30-fold or 5-fold column volumes, respectively. Fractions of 6 ml were collected and analyzed for purity by SDS-PAGE. Fractions containing homogeneous QC were combined and concentrated by ultrafiltration. For long-term storage (-20°C), glycerol was added at 50% of the final concentration. Proteins were quantified according to the method of Bradford or Gill and von Hippel (Bradford, MM 1976 Anal Biochem 72, 248-254; Gill, SC and von Hippel, PH 1989 Anal Biochem 182, 319-326.).

In E. coli QC Expression and purification of

The construct encoding QC was transformed into M15 cells (Qiagen) and incubated at 37° C. on a selective LB agar plate. Protein expression was performed at room temperature in LB medium containing 1% glucose and 1% ethanol. When the culture _{reached an OD 600} of about 0.8, expression was induced with 0.1 mM IPTG overnight. After one cycle of freezing and thawing, the cells were hemolyzed by treatment with 50 mM phosphate buffer containing 300 mM NaCl and 2 mM histidine, 2.5 mg/ml lysozyme at pH 8.0 at 4° C. for 30 minutes. Two additional filtration steps in which the solution was purified by centrifugation at 37000xg for 30 minutes at 4°C, followed by filtration by applying a glass frit (DNA separation) to apply a cellulose filter to the crude and purified precipitates. Was carried out. The supernatant (about 500 ml) was applied on a Ni ^{2 +} -affinity column (1.6 x 20 cm) at a flow rate of 1 ml/min. QC was eluted with 50 mM phosphate buffer containing 150 mM NaCl and 100 mM histidine. The QC-containing fraction was concentrated by ultrafiltration.

From papaya latex QC Tablets of

QCs were prepared from papaya latex with a modified version of the previously reported method (Zerhouni, S. et al. 1989 Biochim Biophys Acta 138, 275-290) using BioCAD 700E (Perseptive Biosystems, Wiesbaden, Germany). 50 g latex was dissolved in water and centrifuged as described in the document above. The protease was inactivated with S-methyl methanethiosulfonate, and the obtained crude extract was dialyzed. After dialysis, the whole supernatant (21 X 2.5 cm i.d.) was loaded onto a SP Sepharose Fast Flow column equilibrated with 100 mM sodium acetate buffer, pH 5.0 (flow rate 3 ml/min). Elution was performed in three steps by increasing the sodium acetate buffer concentration at a flow rate of 2 ml/min. The first step was a linear gradient from 0.1 to 0.5 M of acetate buffer in 0.5 column volume. The second step was to increase the buffer concentration linearly from 0.5 to 0.68 M at 4 times the column volume. In the final elution step, one column volume of 0.85 M buffer was applied. Fractions (6 ml) containing the highest enzymatic activity were combined. The concentration and buffer solution were changed to 0.02 M Tris/HCl, pH 8.0 through ultrafiltration (Amicon; cut-off molecular mass of the membrane 10 kDa).

Ammonium sulfate was added to a final concentration of 2 M to the concentrated papaya enzyme obtained from the ion exchange chromatography step. This solution was applied onto a Butyl Sepharose 4 Fast Flow column equilibrated with 2 M ammonium sulfate, 0.02 M Tris/HCl, pH 8.0 (21 X 2.5 cm id) (flow rate 1.3 ml/min). It was eluted in three steps while lowering the concentration of ammonium sulfate. In the first step, a linear gradient from 2 to 0.6 M ammonium sulfate, 0.02 M Tris/HCl, pH 8.0 was applied to a 0.5 column volume at a flow rate of 1.3 ml/min. In the second step, a linear gradient of 0.6 to 0 M ammonium sulfate, 0.02 M Tris/HCl, pH 8.0 was applied at a flow rate of 1.5 ml/min in a 5-fold column volume. The final elution step was performed by applying 0.02 M Tris/HCl, pH 8.0 to twice the column volume at a flow rate of 1.5 ml/min. All fractions containing QC activity were combined and concentrated by ultrafiltration. The obtained homogeneous QC was stored at -70 °C. The final protein concentration was determined by comparison with a standard curve obtained with bovine serum albumin using the Bradford method.

Example 2 : Glutaminyl Cyclase activity assay

Fluorescence spectrometry ( Fluorometric assays )

All measurements were performed on a microplate (Perkin Elmer) at 30° C. with BioAssay Reader HTS-7000Plus. QC activity was evaluated fluorescence photometrically using H-Gln-βNA. Samples of 0.25 U pyroglutamyl aminopeptidase (Unizyme, Horsholm, Denmark) and QC in 0.2 M Tris/HCl containing 20 mM EDTA, pH 8.0 in a final volume of 250 μl. Consisting of properly diluted partitions. The excitation/emission wavelength was 320/410 nm. The assay reaction was initiated with the addition of glutaminyl cyclase. QC activity was determined from the standard curve of β-naphthylamine under assay conditions. One unit is defined as the amount of QC that catalyzes the formation of 1 μmol pGlu-βNA from H-Gln-βNA per minute under set conditions.

In the second fluorescence spectrophotometry, QC activity was determined using H-Gln-AMC as a substrate. The reaction was carried out at 30° C. using a NOVOStar reader (BMG labtechnologies) on microplates. Samples are in 250 μl final volume, appropriate dilution of various concentrations of fluorogenic substrate, 0.05 M Tris/HCl containing 5 mM EDTA, 0.1 U pyroglutamyl aminopeptidase (Qiagen) and QC in pH 8.0. It consisted of the distribution. The excitation/emission wavelength was 380/460 nm. The assay reaction was initiated with the addition of glutaminyl cyclase. QC activity was determined from the standard curve of 7-amino-4-methylcoumarin under assay conditions. Epidemiologic data were evaluated using GraFit software.

QC of Spectrophotometric Analysis method

Epidemiologic parameters were determined for most QC substrates using this novel assay. QC activity was spectrophotometrically analyzed using a continuous method derived by applying a previous discontinuous assay (Bateman, RCJ 1989 J Neurosci Methods 30,23-28) using glutamate dehydrogenase as a coenzyme. . Samples consisted of each QC substrate, 0.3 mM NADH, 14 mM α-ketoglutaric acid and 30 U/ml glutamate dehydrogenase, in a final volume of 250 μl. The reaction was initiated with the addition of QC, and the absorbance decrease was measured and observed at 340 nm for 8 to 15 minutes. Typical product formation over time is shown in FIG. 1.

Initial rates were evaluated and enzyme activity was determined from a standard curve of ammonia under assay conditions. All samples were measured at 30° C. using a SPECTRAFluor Plus or Sunrise (both from TECAN) reader on microplates. Mechanistic parameters were evaluated using GraFit software.

Inhibitor assay

For the inhibitor test, the sample composition was as described above except that the putative inhibitory compound was added. Rapid QC- inhibition test samples comprised a substrate concentration at 1 K _M of each inhibitor and 4 mM. In order to study the inhibition in detail and _{determine the K i} -value, the effect of the inhibitor on the coenzyme was first investigated. In all cases, no effect was detected in either enzyme, so a reliable determination of QC inhibition could be made. Inhibition constants were evaluated using GraFit software to fit the progression curve to the general formula for competitive inhibition.

Example 3: MALDI - TOF Mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry was performed using a Hewlett-Packard G2025 LD-TOF system equipped with a linear time-of-flight analyzer. The equipment was equipped with a 337 nm nitrogen laser, a powerful source (5 kV) and a 1.0 m flight tube. Detector operation was in positive ion mode and signals were recorded and filtered using a LeCroy 9350M digital storage oscilloscope connected to a personal computer. Samples (5 μl) were mixed with an equal volume of matrix solution. For the matrix solution, 30 mg 2',6'-dihydroxyacetophenone (Aldrich) and 44 mg diammonium hydrogen citrate (Fluka) were added to 1 ml acetonitrile/0.1% TFA-containing water (1/1, v /v), prepared by dissolving in DHAP/DAHC was used. A small volume (=1 μl) of the matrix-analyte-mixture was transferred to the probe tip and immediately evaporated in a vacuum chamber (Hewlett-Packard G2024A sample prep accessory) to rapidly and uniformly crystallize the sample.

For ^{long-term testing of Glu 1} -cyclization, Aβ-inducing peptides were incubated at 30° C. in 100 μl 0.1 M sodium acetate buffer, pH 5.2 or 0.1 M Bis-Tris buffer, pH 6.5. The peptide was applied at a concentration of 0.5mM [Aβ3-11a] or 0.15mM [Aβ3-21a], and 0.2 U QC was added throughout 24 hours. For Aβ(3-21)a, the analyte contained 1% DMSO. At different times, samples were removed from the assay tube, and peptides were extracted using ZipTips (Millipore) according to the manufacturer's instructions, mixed with matrix solution (1:1 v/v), and mass spectra recorded. The negative control did not contain either QC or heat inactivating enzyme. For the inhibitor study, except that the inhibitory compound was added (5 mM benzimidazole or 2 mM 1,10-phenanthroline), the sample composition as described above was used.

Example 4: pH Dependence

The pH-dependence of the catalysis of human and papaya QC was investigated under first order rate conditions, reflecting the effect of proton concentration on the _{specificity constant k cat} /K _M. To this end, a linked enzyme assay using pyroglutamyl aminopeptidase as a coenzyme and Gln-βNA as a substrate was used. Pyroglutamyl aminopeptidase has been shown to be active and stable between pH 5.5-8.5 (Tsuru, D. et al. 1978 J Biochem (Tokyo) 84, 467-476). Thus, this assay makes it possible to study QC catalysis in this pH-range. As shown in Fig. 2, the obtained velocity curve fits a typical bell-shaped curve. Human QC has a very narrow pH-dependence, which is optimal at about pH 7.8-8.0. The rate tended to decrease with more basic pH. This was in contrast to the velocity curve (Fig. 2, inset) observed with Papaya QC, which does not lose activity until pH 8.5. However, both enzymes had optimal specificity at pH 8. _{As a result of evaluating the curves, it was surprisingly found that the same pK a} -values were shown in the acid ranges of 7.17±0.02 and 7.15±0.02, respectively, for human and papaya QCs.

The decrease in human QC activity at the basic pH-value is apparently due to the separation of groups with _{a pK a of about 8.5.} In the case of Papaya QC, there are no excessive data points in the basic pH-range allowing reliable determination of the _{second pK a value.} This is supported by fitting the data to a single segregation model, and when compared to fitting the data to a double segregation model, it results in approximately the same pK _a -value (pK _a 7.13±0.03). This indicates that both pK _a -values are quite apart.

pH stability

Plant and animal enzymes were incubated at 30° C. for 30 minutes at different pH values between pH 4 and 10 to investigate the stability of glutaminyl cyclase. Then, the QC activity was determined under standard conditions. The results are shown in FIG. 3.

QCs from papaya latex were stable in the pH-range studied and did not tend to be apparently unstable in the acidic or basic range. In contrast, human QCs only showed comparable stability in the pH-range between 7 and 8.5, but were significantly unstable at pH values above pH 8.5 and below pH 6. Thus, a range near pH 8 appears to be optimal for plant and human QC activity and stability, and appears to be an appropriate pH-value for performing substrate specificity comparisons of QCs.

Example 5: QC The substrate specificity of

Spectroscopic method

Continuous spectroscopy was performed as described in Example 2. Thus, the decrease in absorbance at 340 nm caused by the ^{release of ammonia and subsequent consumption of NADH/H +} as it is formed from α-ketoglutaric acid to glutamate reflects the QC activity. 1, a linear progression curve was observed, and there was a linear relationship between the measured activity and the QC concentration. Furthermore, the mechanical parameters obtained for H-Gln-Gln-OH using the continuous assay (Table 1) were quite consistent with those obtained using the discontinuous method (K _M = 175 ± 18 μM, k _cat = 21.3 ± 0.6). s ^-1 ). For the conversion of substrates H-Gln-Ala-OH, H-Gln-Glu-OH, H-Gln-Gln-OH, H-Gln-OtBu and H-Gln-NH _{2 by papaya QC shown in Table 1} The mechanical parameters corresponded very well to those determined using the direct method at pH 8.8 and 37° C. (Gololobov, MY et al. 1996 Biol Chem Hoppe Seyler 377, 395-398). Therefore, it is very clear that the novel continuous assay provides reliable results.

D-, tree- and Dipeptide -Substitute ( surrogate )

Using the novel serial assay described above, about 30 compounds were tested as potential substrates for C. papaya and human QC. The results are shown in Table 5. By comparing the specificities, it was shown that almost all short peptide substrates were converted more effectively by papaya QC compared to human enzymes. Interestingly, for both enzymes, the substrate with a large hydrophobic residue at the second position, compared to other tripeptides, is H-Gln-Tyr-Ala-OH, H-Gln-Phe-Ala-NH ₂ and H- As indicated by the specificity of Gln-Trp-Ala-NH ₂ or by the reactivity of the chromogenic substrates H-Gln-AMC, H-Gln-βNA and H-Gln-Tyr-OH compared to the dipeptide substrate, the most potent It was. For papaya QC, this finding is consistent with previous results that specificity is related to the size of the second amino acid residue (Gololobov, MY et al. 1996 Biol Chem Hoppe Seyler 377,395-398).

The only striking difference in the specificity of plant and animal QCs was observed for H-Gln-OtBu. Whereas the ester was converted by papaya QC, which has a similar specificity compared to the dipeptide substrate, it was converted slowly to about first order size by human QC.

Oligopeptide

In addition to several dipeptides and tripeptides, many oligopeptides were tested upon conversion by papaya and human QC (Table 6). Interestingly, the overall difference in specificity between human and plant QCs for a set of tetrapeptides was not as great as observed for dipeptide and tripeptide substrates. This indicates that the amino acids at the 3rd and 4th positions still influence the dynamic behavior of human QCs, especially. However, exceptionally, peptides having a proline residue at the second amino acid position showed a markedly reduced k _cat /K _M value in _{a set of tetrapeptides of the construct H-Gln-X aa} -Tyr-Phe-NH _{2 (} Table 6). The decrease in specificity was further aggravated for human QC, which resulted in an approximately 8-fold difference in _{k cat} /K _{M -values compared to papaya QC.}

Compared to other tetrapeptides, by specificity for H-Gln-Arg-Tyr-Phe-NH ₂ , H-Gln-Arg-Tyr-Phe-NH ₂ and H-Gln-Lys-Arg-Leu-NH ₂ As can be seen, it was found that human QC exhibited slightly reduced specificity in the conversion of the substrate with the positively charged amino acid C-terminus of glutamine. Obviously, the reduced specificity was mainly due to the smaller number of conversions. This effect was not present on plant enzymes.

Epidemiologic evaluation of the peptide substrates of human and papaya QC temperament Human QC Papaya QC K _M (μM) k _cat
(s ^-1 ) K _i ^* (mM) k _cat / K _M
( mM ^-One s ^-One ) K _M (μM) k _cat
(S ^-1 ) K _i ^*
(mM) k _cat / K _M
( mM ^-One s ^-One ) H-Gln-OH n.r. n.r. n.d. n.r. n.d. n.d. n.d. 0.23±0.1 H-Gln-AMC 54±2 5.3±0.1 n.d. 98±2 42±1 39.4±0.4 n.d. 938±13 H-Gln-βNA 70±3 20.6±0.5 1.21±
0.07 294±6 38±3 51.4±1.4 1.20±0.08 1353±70 H-Gln-OtBu 1235±74 6.7±0.2 n.i. 5.4±0.2 223±9 49.4±0.6 n.i. 222±6 H-Gin-NH ₂ 409±40 12.8±0.5 n.i. 31±2 433±13 44.8±0.4 n.i. 103±2 H-Gin-Gly-OH 247±10 13.2±0.2 n.i. 53±1 641±20 45.8±0.4 n.i. 71±2 H-Gln-Ala-OH 232±5 57.2±0.4 n.i. 247±4 158±8 69.8±1.0 n.i. 442±16 H-Gln-Gln-OH 148±5 20.7±0.2 n.i. 140±2 44±3 43.2±0.7 n.i. 982±51 H-Gln-Glu-OH 359±10 24.7±0.2 n.i. 58±1 106±5 50.3±0.6 n.i. 475±17 H-Gln-Val-OH 196±5 17.2±0.1 n.i. 88±2 n.d. n.d. n.i. n.d. H-Gln-Tyr-OH 211±5 94±1 n.i. 446±6 n.d. n.d. n.i. n.d. H-Gln-Gln-Tyr-NH ₂ 79±2 45.1±0.4 n.i. 524±8 103±4 53.6±0.7 n.i. 520±13 H-Gln-Gly-Pro-OH 130±5 25.3±0.2 n.i. 195±7 333±15 41.7±0.5 n.i. 125±4 H-Gln-Tyr-Ala-OH 101±4 125±1 n.i. 930±27 63±3 104.0±1.0 n.i. 1650±63 H-Gln-Phe-Ala-NH ₂ 69±3 109±1 n.i. 1811±64 111±5 132.1±0.6 n.i. 1190±48 H-Gln-Trp-Ala-NH ₂ 50±2 47.0±0.7 n.i. 940±24 78±5 151.8±2.6 n.i. 1946±91 H-Gln-Arg-Gly-Ile-NH ₂ 143±4 33.5±0.4 n.i. 234±4 123±10 49.2±1.7 n.i. 400±19 H-Gln-Asn-Gly-Ile-NH ₂ 172±5 56.6±0.5 n.i. 329±7 153±9 51.4±0.9 n.i. 336±14 H-Gln-Ser-Tyr-Phe-NH ₂ 55±3 52.8±0.8 n.i. 960±38 135±6 64.9±1.0 n.i. 481±14 H-Gln-Arg-Tyr-Phe-NH ₂ 55±2 29.6±0.3 n.i. 538±14 124±6 48.9±0.7 n.i. 394±13 H-Gln-Pro-Tyr-Phe-NH ₂ 1889±
152 31.7±1.2 n.i. 17±1 149±14 18.8±0.6 n.i. 126±8 H-Gln-His-Tyr-Phe-NH ₂ 68±3 55.4±0.7 n.i. 815±26 92±7 75.9±1.4 n.i. 825±48 H-Gln-Gln-Tyr-Phe-NH ₂ 41±2 41.4±0.4 n.i. 1010±40 45±2 52.9±0.7 n.i. 1176±37 H-Gln-Glu-Tyr-Phe-NH ₂ 47±4 46±1 n.i. 979±62 100±4 54.6±0.6 n.i. 546±16 H-Gln-Glu-Ala-Ala-NH ₂ 77±4 46±1 n.i. 597±18 102±4 53.7±0.6 n.i. 526±15 H-Gln-Glu-Tyr-Ala-NH ₂ 69±2 42.1±0.4 n.i. 610±12 113±5 44.7±0.5 n.i. 396±13 H-Gln-Glu-Ala-Phe-NH ₂ 39±3 39±1 n.i. 1000±51 81±3 48.5±0.45 n.i. 599±17 H-Gln-Glu-Asp-Leu-NH ₂ 55±2 45.8±0.5 n.i. 833±21 107±6 58.5±0.4 n.i. 547±27 H-Gln-Lys-Arg-Leu-NH ₂ 54±3 33.4±0.5 n.i. 619±25 118±6 48.2±0.8 n.i. 408±14

(Regarding substrate inhibition, nr, non-reactive; ni, no inhibition; nd, undecided; *)

The results obtained with the tetrapeptide also led to another conclusion. As already pointed out, papaya QC showed higher selectivity for dipeptide. For some tetrapeptides, however, for a set of peptides containing glutamate at the second amino acid position, a higher specificity constant was assigned to human QC, as shown in Figure 4 which provides a graph of the data given in Table 6. Observed. Furthermore, as the chain length increased from di- to tetrapeptide, the selectivity of human QC increased, contrary to the results obtained with papaya QC. In addition, the highest selectivity of human QC was recorded for peptides containing large hydrophobic residues at the 3rd and 4th amino acid positions, indicating a hydrophobic interaction with the enzyme. Comparing the kinetic parameters for each peptide, the change appears to be primarily _{due to the lower K M} -value, revealing that the turnover number for the conversion of the peptide is similar. Thus, it is believed that the higher selectivity of human QCs for longer peptides is a result of the more robust binding of the more hydrophobic substrate to the enzyme.

The differences between human and plant QCs observed with peptides containing hydrophobic amino acids at the 3rd and 4th positions are also H-Gln-Arg-Gly-Ile-NH ₂ and H-Gln-Arg-Tyr-Phe-NH ₂ Or by comparing the specificity constants of the enzymes towards H-Gln-Gln-OH and H-Gln-Gln-Tyr-Phe-OH.

Human QC was also found to be more selective for cognate substrates containing the N-terminal Gln (Table 7). While the selectivity of human QC increases with substrate length, there was no such tendency for papaya QC. Since human QCs are less specific for peptides containing Ser residues in the sequence, the nature of the side chains also appeared to be important (Table 6).

Effect of substrate length on human and papaya QC activity temperament Human QC Papaya QC K _M
(μM) k _cat (S ^-1 ) k _cat / K _M
( mM ^-One S ^-One ) K _M (μM) k _cat (S ^-1 ) k _cat / K _M
mM ^-One S ^-One ) H-Gln-Ala-NH ₂ 155±9 40.1±0.9 259±9 212±21 62.8±3.0 296±15 H-Gln-Ala-Ala-NH ₂ 87±3 76.3±0.7 877±22 164±6 83.2±1.0 507±12 H-Gln-Ala-Ala-Ala-Ala-NH ₂ 65±3 60.5±0.7 1174±43 197±8 74.6±1.0 379±10 H-Gln-Ala-Ala-Ser-Ala-Ala-NH ₂ 79±6 55.3±1.6 700±33 216±6 78.5±1.0 363±5

Effect of ionic strength on catalysis

Another parameter investigated for the effect on substrate specificity was ionic strength. To this end, the kinetic parameters for cyclization of several substrates were determined in the presence and absence of 0.5 M KCl (Tables 8 and 9). Surprisingly, the selectivity for substrates with uncharged backbones did not change significantly with the addition of salts for QCs from papaya latex and human QCs. The specificity constants of human QC for H-Gln-Ala-OH and H-Gln-Glu-OH, however, decreased with the addition of KCl. As indicated by the individual mechanical parameters, this effect is due to the increasing K _M and only slightly decreasing k _cat -values. In the case of Papaya QC, this effect was not detected for any of the variables. This effect does not appear to be due to the negatively charged substrate, since uncharged parameters were found for the negatively charged peptide H-Gln-Glu-Asp-Leu-NH ₂ . An interesting effect of the salt addition was found for the positively charged substrates H-Gln-Arg-Gly-Ile-NH ₂ and H-Gln-Lys-Arg-Leu-NH ₂ . For plant and human QC, the positive effect on catalysis was determined primarily due to the smaller K _M values and slightly larger turnover numbers.

Effect of ionic strength on the catalysis of Papaya QC temperament 0.05 M Tricine-NaOH, pH 8.0 0.05 M Tricine-NaOH, pH 8.0, 0.5 M KCl






papaya
QC K _M (mM) k _cat
(s ^-1 ) k _cat /K _M
(mM ^-1 s ^-1 ) Ki(mM) K _M (mM) k _cat
(s ^-1 ) k _cat /K _M
(mM ^-1 s ^-1 ) Ki(mM) H-Gln-NH ₂ 0.434
±0.015 43.4
±0.4 100±3 n.i. 0.446
±0.010 45.2
±0.3 101±2 n.i. H-Gln-βNA 0.036
±0.002 48.8
±1.0 1356±50 1.14
±0.05 0.032
±0.002 47.2
±0.8 1475±70 1.33
±0.07 H-Gln-Ala-OH 0.137
±0.007 69.7
±.9 509±19 n.i. 0.143
±0.005 68.1
±0.6 480±12 n.i. H-Gln-Glu-OH 0.098
±0.005 45.0
±0.5 459±18 n.i. 0.094
±0.003 44.4
±0.3 472±12 n.i. H-Gln-Trp-Ala-NH ₂ 0.079
±0.005 138±3 1747±73 n.i. 0.072
±0.004 133±3 1847±61 n.i. H-Gln-Arg-Gly-Ile-NH ₂ 0.106
±0.008 52.9
±1.2 499±26 n.i. 0.065
±0.005 48.4
±1.0 745±42 n.i. H-Gln-Lys-Arg-Leu-NH ₂ 0.102
±0.007 50±1 493±22 n.i. 0.053
±0.002 58.1
±0.7 1096±28 n.i. H-Gln-Glu-Asp-Leu-NH ₂ 0.109
±0.005 52.4
±0.7 481±16 n.i. 0.094
±0.003 53.6
±0.5 570±13 n.i.

Effect of ionic strength on the catalysis of human QC 0.05 M Tris-HCl, pH 8.0 0.05 M Tris-HCl, pH 8.0,
0.5 M KCl






Human QC Km(mM) Kca H-Gln-NH ₂ 0.442
±0.030 12.8
±0.3 29±1 n.i. 0.401
±0.014 12.2
±0.1 30±1 n.i. H-Gln-βNA 0.076
±0.004 21.7
±0.5 285±8 1.39
±0.08 0.063
±0.003 20.0
±0.4 318±9 0.97
±0.04 H-Gln-Ala-OH 0.269
±0.007 54.4
±0.5 202±3 n.i. 0.357
±0.012 47.6
±0.6 133±3 n.i. H-Gln-Glu-OH 0.373
±0.015 21.4
±0.3 57±2 n.i. 0.607
±0.036 18.9
±0.5 31±1 n.i. H-Gln-Trp-Ala-NH ₂ 0.054
±0.003 50.8
±0.6 941±41 n.i. 0.056
±0.002 50.0
±0.4 893±25 n.i. H-Gln-Arg-Gly-Ile-NH ₂ 0.166
±0.013 31±1 187±9 n.i. 0.091
±0.005 29.8
±0.5 327±12 n.i. H-Gln-Lys-Arg-Leu-NH ₂ 0.051
±0.003 29.4
±0.5 577±24 n.i. 0.034
±0.001 31.6
±0.3 929±19 n.i. H-Gln-Glu-Asp-Leu-NH ₂ 0.060
±0.002 46.6
±0.5 777±18 n.i. 0.061
±0.002 45.6
±0.5 748±16 n.i.

Physiological substrate

In earlier studies, ^{the conversion of [Gln 1} ]-TRH and [Gln ¹ ]-GnRH by QC was already seen in QCs obtained from the pituitary gland of cattle and pigs (Busby, WHJ et al. 1987 J Biol Chem 262, 8532-8536). ; Fischer, WH and Spiess, J. 1987 Proc Natl Acad Sci USA 84, 3628-3632). In addition to these already investigated pituitary hormone, the three potential physiological substrates of human QC, name Gln ¹ - gastrin, Gln ¹ - neurotensin were synthesized and tested upon conversion Gln ¹ -FPP. The kinetic parameters for their conversion are listed in Table 1. Interestingly, the glutaminyl peptides are each converted to pyroglutamyl peptides, which are their size, i.e., the largest peptide with 17 amino acids, followed by pro-gastrin, pro-neurotensin, pro-GnRH, pro-TRH and pro. The specificity constant increased depending on the size followed by -FPP. These findings corresponded to data obtained from synthetic peptides.

Surprisingly, longer substrates are also converted by plant enzymes with higher selectivity, which partially contradicted the findings in shorter oligopeptides. Perhaps, far from the active site, there may have been a second binding interaction between the substrate and the enzyme.

Containing modified amino acids Peptide

To further investigate the specificity and selectivity of QC, a peptide containing an N-terminal glutaminyl residue or an amino acid modified at the second position was synthesized. The conversion of these peptides was qualitatively analyzed by MALDI-TOF mass spectrometry (see Example 3). Due to the cyclization of the glutaminyl residues or analogs thereof, respectively, the difference in mass of the catalytic substrate and product was detected. The release of 1 mole of ammonia per mole of substrate was also analyzed qualitatively by spectroscopic analysis.

H- Gln - Lys ( Gln ) -Arg - Leu - Ala - NH ₂ . These N-terminal branched peptides, comprising two glutaminyl residues at the N-terminus bonded to a lysyl residue through a peptide- and partial iso-peptide bond, are in the same way human (Figure 5) and Papaya QC (mi Time). Both glutaminyl residues were converted to pyroglutamic acid, without any detectable preference for distinct residues, as indicated by the matched substrate conversion (Figure 5). Thus, the selectivity of QC for differently bound glutaminyl residues was not fundamentally different.

H- Gln (NMe) - Phe - Lys - Ala - Glu - NH 2. The methylated glutaminyl residues were converted only to pyroglutamyl residues by Papaya QC (FIG. 6 ). In addition, no inhibition of human QC by the peptide was detected, indicating that the methylated residue was not recognized by human QC.

H- Glu ( OMe )-β NA and H- Glu- β NA . None of these compounds were converted by papaya or human QC. These fluorescent substrates were analyzed by fluorescence spectrophotometry, using pyroglutamyl aminopeptidase as a coenzyme. The O-methylated glutamate residues, however, were shown to be very unstable, showing a tendency of non-enzymatically catalyzed cyclization in both Tris and Trisine buffers. Furthermore, both QC activity against H-Gln-AMC as a substrate is the longer peptides H-Glu(OMe)-Phe-Lys-Arg-Leu-Ala-NH ₂ or H-Glu-Phe-Lys-Arg- It was not inhibited by Leu-Ala-NH ₂ , indicating that glutamic acid or derivatives were not recognized by both QC forms. Furthermore, the results indicate that the negative charge of the glutamic acid residue is not the only reason to reject the peptide from the active site.

H- Gln -cyclo (Nε- Lys - Arg - Pro - Ala - Gly - Phe ) . The conversion of H-Gln-cyclo (Nε-Lys-Arg-Pro-Ala-Gly-Phe) with partial iso-peptide bonds inside the molecule was quantitatively analyzed, and 240±14 μM for human and papaya QC, respectively. _{And a K M} -value of 133±5 μM was shown. Due to the higher turnover number of conversions by papaya QC (49.4±0.6 s ^-1 ) compared to human QC (22.8±0.6 s ^-1 ), plant enzymes are about 4 times higher than human QCs k _cat /K _M- It showed a ^1-value 372 ± 9 mM ^-1 min. Therefore, in the case of papaya QC, the specificity constant was only slightly smaller than that of a substrate having a similar size such as H-Gln-Ala-Ala-Ser-Ala-Ala-NH _{2. The k cat} /K _M value for human QC, however, was found to have 95±3 mM ^-1 s ^{- 1} , which was about a smaller first order size compared to similarly sized substrates (Table 5).

H-β homo Gln - Phe - Lys - Arg - Leu - Ala - NH ₂ . The N-terminal β-homoglutaminyl residue was converted to a 5-membered lactam ring by catalysis of human and papaya QC, respectively. Concurrent ammonia emissions were analyzed spectroscopically and by MALDI-TOF analysis as described above. When QC was removed or heated, no ammonia emissions were detected, indicating a specific catalysis of cyclization. Interestingly, the QCs from C. papaya (K _M = 3.1±0.3 mM, k _cat = 4.0±0.4 S ^-1 ) and human (K _M = 2.5±0.2 mM, k _cat = 3.5±0.1 S ^-1 ) are These peptide conversions were catalyzed with _{approximately the same k cat} /K _M values of 1.4±0.1 and 1.3±0.1 mM ^-1 s ^-1 , respectively. Therefore, the cyclization of the β-homoglutamine residue was catalyzed with an efficiency reduced by about 1000 times compared to a peptide of a similar size containing a glutaminyl residue at the N-terminus. This indicates that the constitution of the α-carbon of the substrate is important, but not essential, for substrate recognition by the QC form. The essential requirement to become a substrate was a γ-amide group and an aprotonated N-terminal amino group at a distance and angle easy to cyclize, which is carried out by the N-terminal glutaminyl and β-homo-glutaminyl residues. It is a requirement.

Example 6: QC Synthesis of substrates

Oligopeptides . Using a peptide synthesizer (Labortec SP650, Bachem, Switzerland), as described above (Schilling, S. et al. 2002 Biochemistry 41, 10849-10857), the peptide was synthesized semi-automatically at a 0.5 mmol scale. Longer peptides were synthesized at 25 μmol scale as described (Manhart, S. et al. 2003 Biochemistry 42,3081-3088) using an automated Symphony Peptide synthesizer (Rainin Instrument Co.). For all peptide couplings, the modified Fmoc-protocol of solid-phase peptide synthesis, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU; Novabiochem) / base (diisopropyl ethylamine or N-methyl-morpholine; Merck) was used, and in the case of difficult conjugation, N-[(dimethylamino)-1H-1,2,3 -Activation of triazolo[4,5-b]pyridin-1-ylmethylene]-N-methylmethaneamminium hexafluorophosphate N-oxide (4,5) (HATU; Applied Biosystems)/diisopropyl ethylamine Used as a reagent. After cleavage from the resin with a cocktail containing trifluoroacetic acid (TFA; Merck), the crude peptide was purified by preparative HPLC using an acid-free solvent to avoid cyclization of further N-terminal glutamine. Preparative HPLC was performed using a linear gradient of acetonitrile (Merck) (5-40% or 65% acetonitrile over 40 minutes) contained in water on a 250-21 Luna RP18 column (Phenomenex). To confirm the peptide purity and identity, analytical HPLC and ESI-MS were used.

Glu ( NH - NH ₂ ) -Ser - Pro - Thr - Ala - NH ₂ . The straight precursor peptide (Fmoc-Glu-Ser-Pro-Thr-Ala-NH ₂ ) was synthesized on Rink amide MBHA resin (Novabiochem) according to the standard Fmoc-procedure (Schilling, S. et al. 2002 Biochemistry 41, 10849-10857). I did. After cleaving the Fmoc-protecting peptide from the resin, the peptide was precipitated with diethyl ether (Merck), filtered and dried. HMBA-AM resin (1.16 mmol/g, Novabiochem) in dichloromethane (DCM, Merck) was used for conjugation of the γ-carboxylic acid group of glutamic acid of the precursor peptide (3 eq.). Dicyclohexylcarbodiimide (DCC, Serva) (4 eq.) and dimethylaminopyridine (DMAP, Aldrich) (0.1 eq) were used as a pairing reagent. After 12 hours, the resin was filtered, washed with DCM and the reaction was repeated. After deprotection of the N-terminal Fmoc-group using a DMF solution containing 20% piperidine (3 x 5 min), the peptide resin was treated with a 5% hydrazine solution (20 ml/g) for 1.5 hours. The resin was filtered and washed with dimethylformamide (DMF, Roth, Germany) and TFA. After evaporation, the crude peptide was precipitated with ether to obtain 76% yield.

H- Gln - Lys ( Gln ) -Arg - Leu - Ala - NH ₂ . Following the standard Fmoc/ ^t Bu-procedure, the linear peptide was transferred to Rink amide MBHA (Schilling, S. et al. 2002 Biochemistry 41, 10849-10857) with Fmoc-Lys(Fmoc)-OH as the second amino acid pairing at the end. Was synthesized using. After deprotection of the two amino protecting groups of lysine using DMF containing 20% piperidine (Merck), 4 eq. Fmoc-Gln(Trt)-OH was paired. The standard cutting procedure yielded 95%.

H- Gln (NMe) - Phe - Lys - Ala - Glu - NH 2. Starting from Fmoc-Glu-OtBu loaded on Fmoc-MI-AM (Novabiochem) resin, Fmoc-Gln(NMe)-OH was synthesized. After inflating with DCM, the resin (0.5 g) was washed with DMF and deprotected with DMF containing 20% piperidine solution. After adding the resin to 5 ml DMF, 5 eq. Fmoc-Glu-OtBu, 5 eq. HATU and 10 eq. DIPEA was added and shaken for 6 hours. After filtration and washing, the product was cut according to standard TFA cleavage conditions. The peptide H-Gln(NMe)-Phe-Lys-Ala-Glu-NH ₂ was synthesized as described (Schilling, S. et al. 2002 Biochemistry 41, 10849-10857). Fmoc-Gln(NMe)-OH was paired with HATU/DIPEA overnight. The standard cleavage procedure yielded the crude peptide in 78% yield.

H- Glu ( OMe )-β -naphthylamide , H- Gln - Val - OH , H- Gln - Tyr - OH . Boc-protected dipeptide was synthesized using isobutyl chlorocarbonate (Merck) according to a standard mixed anhydride procedure. The C-terminal methyl esters Boc-Gln-Tyr-OMe and Boc-Gln-Val-OMe were saponified using dioxane containing 1 N NaOH. The Boc-protected peptide was deprotected for 10 minutes with HCl/dioxane solution. After evaporation, the residue was crystallized using several solvents to give a solid compound of 60 to 70%.

H- Gln -cyclo (Nε- Lys - Arg - Pro - Ala - Gly - Phe ) . The linear precursor Boc-Gln(Trt)-Lys-Arg(Pmc)-Ala-Gly-Phe-OH was synthesized on acid-sensitive 2-chlorotrityl resin. Pairing was performed according to the standard protocol of ^{the Fmoc/t} Bu-strategy using Fmoc-Lys(Mtt)-OH. After cutting with DCM containing 3% TFA solution (10 times, 5 minutes), the solution was neutralized with methanol (MeOH; Merck) containing 10% pyridine (Merck), washed 3 times with DCM and MeOH, and a volume of 5% By evaporation, the crude peptide was precipitated with ice-cold water. Then, the crude peptide was cyclized using DCC/N-hydroxybenzotriazole (HOBt; Aldrich) activation. The crude peptide was dissolved in dry dichloromethane (0.2 mmol/50 ml), and 0.2 mmol N-methylmorpholine and 0.4 mmol 1-hydroxybenzotriazole were added. This solution was added dropwise to 250 ml dichloromethane solution containing 0.4 mmol dicyclohexylcarbodiimide at 0°C. The reaction was completed while stirring at room temperature overnight. After filtration of N,N'-dicyclohexylurea, the solvent was evaporated off. The residue was dissolved in ethyl acetate and washed several times with a saturated solution of _{1N HCl, NaHCO 3 and water.} The solution was dried over anhydrous Na ₂ SO ₄ , filtered and evaporated to dryness in vacuo.

Example 7: QC Effector Characterization

Imidazole derivative

Imidazole and benzimidazole derivatives having substituents at different positions of the 5-membered ring were tested as inhibitors of QC (Table 3). The composition of the number refers to the imidazole ring. The method applied is described in Example 2.

C-4(5) and C-4,5 derivatives. Compounds having substituents at either or both of the constitutively equivalent 4- or 5-positions of the imidazole ring showed reduced efficacy against inhibition of human QC. However, exceptionally, N-ω-acetylated histamine has proven to be one of the most potent inhibitory compounds. Small substituents at this position had little effect on binding compared to imidazole, as indicated by the similar inhibitory constant of 5-hydroxymethyl-4-methyl-imidazole. Larger and larger groups attached to these sites reduced or eliminated enzymatic compound binding. Several other substituents tested are known to exert negative inductive or mesomeric effects, which can reduce the electron density in the imidazole ring, and also weaken the binding constant. _{Differences in the K i} -values of L-histidine and histidineamide also showed some effect of charge on binding. Evidence for electrostatic rejection of charged substrates has already been shown in substrate specificity studies, ie glutamineamide is readily converted by human QC, but no reactivity was observed for free glutamine as a substrate.

C-2 derivatives. All of the tested derivatives inhibited QC more weakly as imidazole. Any substitution larger than a proton prevented proper QC-binding. Due to the methyl group in only 2-methyl-benzimidazole, the inhibition constant decreased to about a first order size. A very similar relationship was shown by comparing the _{K i} -values for benzimidazole and 2-amino-benzimidazole. In addition, the results indicated that the effect was independent of the electronic change.

N-1 derivatives. Among the imidazole derivatives tested for human QC inhibition, _{most compounds with improved K i} -values compared to imidazole showed an alteration at one nitrogen atom. These compounds also include one of the most effective QC inhibitors, 1-benzylimidazole. Interestingly, minor changes to these structures resulted in the loss of inhibitory properties, as can be seen for 1-benzoylimidazole and phenylimidazole, which are inactive under experimental conditions. Also in this case, the observed change did not appear to be caused only by the reduced electron density of the imidazole ring due to the negative resonance (mesomeric) effect of the phenyl group, which also showed that the large trimethyl-silyl group exhibiting a positive induction effect compared to other residues. This was because it showed reduced bonding. Interestingly, one of the less effective compounds of this group was 1-aminopropyl-imidazole. The small potency of this compound is caused by the basic amino group, because spatially similar compounds, 1-methylimidazole and 1-vinylimidazole, show improved binding to the active site. Thus, the positively charged amino group exhibited a smaller K _i -value, which was confirmed by comparing _{the K i} -value of N-ω-acetylated histamine (Table 3) and histamine (Table 4).

Effects of 3,4 and 3,5 derivatization . It has been shown that imidazole derivatives containing substituents at position 4(5) or both have limited efficiency in binding to enzymes. The effect of specific substituents was clarified by comparing the inhibitory constants of the two intermediates, 3-methyl-4-histamine and 3-methyl-5-histamine, in the biodegradation of L-histamine and histamine (Table 4). _{L-histamine exhibited a small K i} value that was about the order of magnitude compared to its acetylated counterpart. Methylation of one nitrogen resulted in a significant improvement in efficacy for 3-methyl-4-histamine. Methylation leading to 3-methyl-5-histamine, however, resulted in a complete loss of inhibitory activity. Thus, the observed effect appeared to be mainly due to the spatial hindrance of the bond due to derivatization of carbon adjacent to basic nitrogen. Presumably, basic nitrogen appeared to play an important role in binding to enzymes.

Example 8: Formation of Aβ(3-40/42) derivative

Measurements were taken of two short N-terminal peptide sequences of Aβ (3-40/42) containing glutamine instead of glutamic acid residue at the third position, Gln ³ -Aβ(1-11) (SEQ ID NO: DAQFRHDSGYE) and Gln ^{3 -Aβ.} It was carried out with (3-11)a. Cleavage by DP IV and cyclization of the N-terminal glutamine residue by QC of the two peptides were tested by MALDI-TOF mass spectrometry. Measurements were carried out using purified DP IV (pork kidney) or raw porcine pituitary crushed product as a QC source and as an enzyme source to measure continuous catalysis.

result

1. The formation of Gln ³ -Aβ (3-11) from the Gln ³ -Aβ1-11a catalyzed by the DP and DP Ⅳ a Ⅳ- inhibitors Val- pyrrolidin the prevention thereof by Id (Val-Pyrr)

DP IV or DP IV-like activity ^{cleaves Gln 3} -Aβ(1-11)a to form Gln ³ -Aβ(3-11)a (Fig. 7). The residue at the third position is exposed by this cleavage and thus alteration by other enzymes, namely QC, has become accessible. As expected, catalysis can be completely prevented by Val-Pyrr (Fig. 8).

^{2. Formation of pGlu 3 -Aβ(3-11)a from Gln 3} -Aβ(3-11)a by catalysis of QC in pituitary pulverization and prevention by 1,10-phenanthroline

Glutaminyl cyclase in the pulverized product of the porcine pituitary gland ^{catalyzes the conversion of Gln 3} -Aβ(3-11)a to [pGlu ³ ]-Aβ(3-11)a (FIG. 9). The formation of [pGlu ³ ]Aβ(3-11)a was inhibited by the addition of 1,10-phenanthroline (FIG. 10).

3. Continuous catalysis of DP IV and QC resulting in the formation of [pGlu ³ ]Aβ(3-11)a and prevention by Val-Pyrr and 1,10-phenanthroline

^{The formation of [pGlu 3} ]Aβ(3-11)a from Gln ³ -Aβ(1-11)a was measured in the pulverized product of the porcine pituitary gland to which DP IV obtained from pig kidney was added, as measured by DP IV and QC. Occurred after the continuous catalysis of (Fig. 11). [pGlu ³ ]Aβ(3-11)a was not formed when QC-inhibitor 1,10-phenanthroline (FIG. 12) or DP IV-inhibitor Val-Pyrr (FIG. 13) was added. The weak appearance of [pGlu ³ ]Aβ(3-11)a was due to aminopeptidase cleavage and subsequent cyclization of glutamine residues, as indicated by the formation of ^{[Gln 3]Aβ(2-11)a.} .

^{4. Formation of [pGlu 3} ]Aβ(3-11)a by catalytic action of aminopeptidase(s) in crude pituitary pulverization

^{Due to the formation of [pGlu 3} ]Aβ(3-11)a, which is not dependent on DP IV catalysis ^{, the decomposition of [Gln 3} ]Aβ(1-11)a in the raw pituitary pulverized product without DP IV was investigated. (Fig. 14). As expected from the data in section 4, [pGlu ³ ]Aβ(3-11)a formation was observed. These data show ^{that degradation of Gln 3} -Aβ(1-11)a can also be catalyzed by aminopeptidase(s), ^{resulting in the formation of [pGlu 3} ]Aβ(3-11)a. Show. Thus, the results indicate that in these tissues, pyroglutamyl formation is the endpoint of N-terminal peptide degradation, further supporting the role of QC in plaque formation.

Example 9: recombinant human QC On by Gln ³ -Aβ(3-11)a; Turnover of (3-21)a and (3-40)

All Gln ³ -Aβ inducing peptides tested were effectively converted to the corresponding pyroglutamyl form by human QC (Tables 8 and 9). Since Gln ³ -Aβ(3-21)a and Gln ³ -Aβ(3-40) have low solubility in aqueous solution, the crystals were carried out in the presence of 1% DMSO. However, ^{due to the better solubility of Gln 3} -Aβ(3-11)a, a kinetic analysis of QC-catalyst turnover in the presence and absence of DMSO was possible (Tables 8 and 9). Taken together, the results of examining Aβ peptide as a QC-substrate with chain lengths of 8, 18 and 37 amino acids (see Table 10) confirmed the observation that human QC-activity increases with the length of its substrate. Accordingly, Gln ¹ - gastrin, Gln ¹ - neurotensin, and Gln ¹ -GnRH are, given the specificity constant, the highest of the QC- substrates. Similarly, the largest QC-substrates investigated to date, Gln ³ -Aβ (3-40) and glucagon, showed high second order rate constants even in the presence of 1% DMSO (449 mM ^-1 s ^-1 and 526 mM ^-1 s ^-1 ) (Table 10).

Interestingly, the kinetic parameters for the conversion of the investigated amyloid peptides did not change drastically with increasing size, suggesting only a moderate effect of the C-terminal portion of Aβ on QC catalysis. Therefore, due to better solubility and experimental handling, smaller fragments of Aβ, Gln ³ -Aβ(1-11)a, Gln ³ -Aβ(3) with respect to the N-terminal aminopeptidase processing of these peptides. It was investigated using -11)a and Aβ(3-11)a.

Epidemiologic parameters for conversion of the N-terminal Gln-containing peptide by recombinant human QC in a buffer containing 1% DMSO peptide Peptide K _M (μM) kcat(s ^-1 ) k _cat /K _M (mM ^-1 s ^-1 ) [Gln ³ ]Aβ3-11a 87±3 ^# 55±1 ^# 632±10 ^# [Gln ³ ]Aβ3-11a 155±4 41.4±0.4 267±4 [Gln ³ ]Aβ3-21a 162±12 62±3 383±10 [Gln ³ ]A3-40 89±10 40±2 449±28 Glucagon (3-29) 19±1 10.0±0.2 526±17

^# Determined in the absence of DMSO

Example 10: recombinant human QC Turnover of Aβ(3-11)a and Aβ(3-21)a by

Incubation of Aβ(3-11)a and Aβ(3-21)a in the presence of QC revealed that, contrary to previous results, glutamate-comprising peptides also act as QC-substrates (Figures 15C and D). . The QC-catalyst formation of [pGlu ³ ]Aβ(3-11)a and [pGlu ³ ]Aβ(3-21)a was investigated at pH 5.2 and 6.5, respectively. If the QC-inhibitor benzimidazole is added to the solution before starting the assay with QC addition, ^{substrate conversion resulting in [pGlu 3} ]Aβ(3-11)a or [pGlu ³ ]Aβ(3-21)a is Was inhibited (Figs. 15E and F). When QC was heated before addition, the formation of pGlu-peptide was negligible (Figs. 15A and B).

Example 11: Papaya QC - Catalytic Gln -β NA And Glu -β NA Of the cyclized pH -Dependency

Papaya QC converted Glu-βNA to a concentration range of up to 2 mM (which was limited by substrate solubility) according to Michaelis-Menten kinetics (FIG. 16 ). A study of the turnover versus substrate concentration plot for QC-catalyzed conversion of Glu-βNA, studied between pH 6.1 and 8.5, showed that for this Glu-substrate, both parameters, K _M and k _cat -It was revealed that it changes in a dependent manner (Fig. 16). This _{was in contrast to the previously described QC-catalyzed glutamine cyclization, where changes in K M} were observed over a given pH range (Gololobov, MY, Song, I., Wang, W., and Bateman, RC (1994) Arch Biochem Biophys 309,300-307).

Subsequently, to study the impact of proton concentration during Glu- and Gln-cyclization, Glu-βNA and Gln-βNA under _{first-order rate law conditions (i.e., substrate concentration well below K M -value).} The pH-dependence of the cyclization of was investigated (Fig. 17). The cyclization of glutamine has a pH-optimality at pH 8.0, which is in contrast to the cyclization of glutamic acid, which has a pH optimum at pH 6.0. While the specificity constant at each optimal pH differed by about 80,000 times, the ratio of QC to EC activity near pH 6.0 was only about 8,000.

Non-enzymatic pGlu-formation from Gln-βNA was investigated at pH 6.0, and after 4 weeks, it was found that the first order rate constant was 1.2*10 ^-7 s ^-1 . However, no pGlu-βNA was formed from Glu-βNA during the same period, which could be estimated that ^{the limiting rate constant for turnover is 1.0*10 -9} s ^-1.

Example 12: enzyme inactivation/reactivation process

Distribution of human QC (0.1-0.5 mg, 1 mg/ml) was mixed with 5 mM 1,10-phenanthroline or 0.05 M Bis-Tris/HCl containing 5 mM dipicolinic acid, 3000 times of pH 6.8. The excess was dialyzed overnight to inactivate. Subsequently, the inactivating agent was carefully removed (3 times, 2000-fold excess dose) by dialysis of the sample against 0.05 M Bis-Tris/HCl containing 1 mM EDTA, pH 6.8. Zn ⁺⁺ , Mn ⁺⁺ , Ni ⁺⁺ , Ca ⁺⁺ , K ⁺ and Co ⁺⁺ ions at a concentration of 1.0, 0.5, 0.25 mM at 0.025 M Bis-Tris containing 0.5 mM EDTA, pH 6.8. Using, a reactivation experiment was performed at room temperature for 15 minutes. To avoid rapid activation by trace metal ions present in the buffer solution, the QC activity assay was performed at 0.05 M Tris/HCl containing 2 mM EDTA, pH 8.0.

Inhibition of porcine QC by 1,10-phenanthroline has already been described (Busby, W. H. J. et al. 1987 J Biol Chem 262, 8532-8536, Bateman, R. C. J. et al. 2001 Biochemistry 40). However, the fact that EDTA was shown to have an activating effect on QC catalysis suggested that inhibition by phenanthroline was not due to metal chelation (Busby, WHJ et al. 1987 J Biol Chem 262, 8532-8536, Bateman). , RCJ et al. 2001 Biochemistry 40). In addition, not only inhibited by 1,10-phenanthroline, human QC catalyzed substrate cyclization was absent in the presence of dipicolinic acid and 8-hydroxyquinoline, other inhibitors of metalloenzymes. These chelators inhibited QC in a competitive and time-dependent manner. That is, it was shown that the initial activity, which was already competitively inhibited, was further reduced after long incubation with the compounds (Figs. 18 and 19). Interestingly, EDTA did not show significant inhibition regardless of incubation time or under any conditions.

Human QC was almost completely inactivated after dialysis in enormous amounts against 5 mM 1,10-phenanthroline or 5 mM dipicolinic acid. After repeated dialysis overnight against a chelator-free buffer solution, the QC activity partially reactivated to 50-60%. However, upon dialysis against a buffer containing 1 mM EDTA, no reactivation was observed.

Nearly complete recovery of QC activity after inactivation _{with dipicolinic acid or 1,10-phenanthroline was achieved by incubating the protein with 0.5 mM ZnSO 4 in the} presence of 0.5 mM EDTA for 10 minutes (Figure 20). Similarly, partial restoration of QC activity was achieved using ^Co++ and Mn ^{++ ions for reactivation.} Even in the presence of 0.25 mM Zn ⁺⁺ , reactivation was possible up to 25% of the original activity. No reactivation was observed when ^Ni++ , Ca ⁺⁺ or K ^{+ ions were applied.} Similarly, QCs that were fully active with these ions had no effect on enzyme activity upon incubation.

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Claims (12)

Glutaminyl cyclase (QC) inhibitors that inhibit the conversion of glutamic acid or glutamine to pyroglutamic acid residues at the N-terminus of Aβ (amyloidβ) 3-40 / 42 and [Gln3] Aβ3-40 / 42 In the pharmaceutical composition for parenteral, digestive tract or oral administration for the treatment of Alzheimer's disease or Down syndrome,
Pharmaceutical composition wherein the glutaminel cyclase (QC) inhibitor is 1,10-phenanthroline or dipicolinic acid. delete The pharmaceutical composition of claim 1, wherein the glutaminel cyclase (QC) inhibitor is used in combination with at least one conventional carrier or excipient or carrier and excipient. The pharmaceutical composition of claim 1, wherein the glutaminel cyclase (QC) inhibitor is a competitive inhibitor. A pharmaceutical composition according to claim 1 for oral administration. delete delete delete delete delete delete delete

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